Human capsaicin receptor and uses thereof

ABSTRACT

This invention provides novel human capsaicin receptors and the nucleotide sequences encoding these receptors. Also provided are vectors encoding these receptors and mammalian and non-mammalian cells expressing these vectors. Further provided are assays for identifying compounds that modulate capsaicin receptors and diagnostic assays for capsaicin receptor polymorphisms and aberrant capsaicin receptor expression.

BACKGROUND OF THE INVENTION

[0001] The sensation of pain can be triggered by any number of physicalor chemical stimuli. In mammals, the peripheral terminals of a group ofspecialized small diameter sensory neurons, termed “nociceptors” mediatethis response to a potentially harmful stimulus.

[0002] In efforts to discover better analgesics for the treatment ofboth acute and chronic pain, and to develop treatments for variousneuropathic pain states, considerable research has been focused on themolecular mechanism of nociception. The response to heat, lowextracellular pH, or capsaicin (the compound responsible for the hotnessof hot peppers) is characterized by the persistent activation ofnociceptors (Bevan and Gepetti, 1994, and Kress and Reeh, 1996). It hasbeen shown that both heat and capsaicin are capable of activating dorsalroot ganglion and trigeminal ganglion neurons via and influx of cations(Oh, et al. 1996, Kirshstein, et al. 1997). Additionally, moderatelyacidic conditions produce this response (Zeilhofer, et al., 1997) andalso potentiate the response of nociceptors to heat and capsaicin(Kress, et al., 1996).

[0003] Capsaicin responses in isolated sensory neurons showdose-dependence and are also evoked by structural analogues of capsaicin(Szolcsanyi and Jancso-Gabor, 1975 and 1976). Resiniferatoxin (RTX), anatural product of Euphorbia plants is a particularly potent activatorof the capsaicin response (Szallasi and Blumberg, 1989). Capsaicin andresiniferatoxin share a common vanilloid moiety, thus the capsaicinreceptor is also termed the vanilloid receptor (VR). The capsaicinresponse is competitively inhibited by another structural analog,capsazepine (Bevan, et al., 1992) and by the non-selective cationchannel blocker ruthenium red (Wood, 1988).

[0004] It was initially postulated that the VR is a non-selective cationchannel with a preference for calcium. Consequently, a ⁴⁵Ca²⁺-uptakeassay using intact rat dorsal root ganglion (DRG) neurons has been usedextensively to characterize structure-activity relations for vanilloids.Specific binding of [³H]RTX provided the first unequivocal proof for theexistence of a VR and has furnished a new, biochemical tool to study VRpharmacology. Such studies, however, have been limited by the lack ofavailability of cloned VR species and sub-types, by the low levels of VRproduced by the few cell types that naturally express such receptors invivo, and by the limited expression levels heretofore obtained usingtransient recombinant expression technologies.

[0005] Interest in characterizing VRs led to the cloning of a functionalrat capsaicin receptor (VR1), from a rat dorsal root ganglion cDNAlibrary (Caterina, et al., 1997). The cDNA for the rat capsaicinreceptor VR1 encodes an 838 amino acid protein (SEQ ID NO:9) with apredicted molecular mass of 95,000 Daltons.

[0006] Sequence analysis suggests that the receptor is composed of a 432amino acid hydrophilic amino terminus that contains a proline-richregion followed by three ankyrin repeat domains, a membrane bound regionthat includes 6 beta-sheet transmembrane domains as well as anadditional membrane-associated region between transmembrane segments 5and 6, and a 154 amino acid carboxy terminus.

[0007] VR1 is activated not only by vanilloids but also by noxious heatand low pH. As predicted, this VR1 is a relatively non-selective cationchannel with a preference for calcium. In Xenopus oocytes expressingVR1, vanilloids evoke inward currents, with RTX being approximately20-fold more potent (EC₅₀=39 nM) than capsaicin (EC₅₀=710 μM). InVR1-transfected mammalian (HEK293) cells, capsaicin induces whole-cellcurrents with a potency of 110 nM. Taken together, these results suggestthat VR1 corresponds to the site in DRG neurons that mediates calciumuptake.

[0008] Homology searches comparing the cloned rat capsaicin receptor VR1to other known ligand gated channels have revealed some relatedreceptors. The most highly homologous protein identified to date is therecently identified rat vanilloid-receptor-like protein 1 (VRL-1)(Caterina, et al. 1999). This protein shares approximately 49% identicalamino acid residues and overall is 66% similar in sequence to the ratcapsaicin receptor, VR1, and is predicted to have a tertiary structurequite similar to that of the capsaicin receptor. While the VRL-1 proteinhas been reported to respond to high temperatures by allowing an influxof cations, it is not a capsaicin receptor, as it is insensitive tocapsaicin and capsaicin analogues.

[0009] Another class of receptors that shows some homology to thecapsaicin receptor is the TRP (transient release potential) family ofputative store-operated calcium channels. (Caterina, et al., 1997) alsoknown as “trp channels”. Members of this family of receptors mediate theentry of extracellular Ca²⁺ in response to the depletion ofintracellular Ca²⁺ stores (Clapham, 1996). The capsaicin receptor, whilemediating the entry of Ca²⁺ and other cations in response to heat, lowextracellular pH and capsaicin and related compounds, does not act as astore-operated calcium channel.

[0010] If vanilloid binding and calcium uptake are always mediated bythe same receptor, a logical prediction would be that ligands mediatingthese two responses should display similar structure-activityrelationships. With regard to DRG neurons expressing native VRs this isclearly not the case: structure-activity analysis of different vanilloidderivatives revealed that the various compounds have distinct potenciesfor receptor binding and for inducing ⁴⁵Ca²⁺-uptake in rat DRG neurons.Although some compounds, such as RTX-amide, bind to VRs and evokecalcium influx with similar potencies, other vanilloids show relativeselectivity for one or the other response. RTX represents one extreme.It is approximately 25-fold more potent for binding (using intact ratDRG neurons the K_(d) was reported to be 40 pM) than for inducingcalcium uptake (EC₅₀=1.0 nM). Capsaicin represents the opposite extreme.It evokes calcium influx with an EC₅₀ of 270 nM but inhibits [³H]RTXbinding with a 10-fold lower affinity of 3 uM. The most straightforwardexplanation appeared to be that RTX binding and calcium uptake detectedtwo distinct classes of VRs. These putative receptors were referred toas R-type (preferentially labeled by RTX) and C-type (displaying ahigher potency for capsaicin) VRs, respectively. This model was furthersupported by the identification of non-neuronal cell lines thatexhibited calcium uptake in response to vanilloid stimulation (implyingthe presence of C-type VRs) but which lacked detectable RTX-specificbinding sites.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1A. Specific binding of [³H]resiniferatoxin to HEK 293 cellstransfected stably with a cDNA encoding the rat VR1 (HEK293/VR1 cells).[³H]Resiniferatoxin displays saturable specific binding to HEK293/VR1cells. Binding data are from a single experiment; points are mean valuesof triplicate determinations; error bars indicate S.E.M. The bindingcurve was generated by a computer fit of the measured values to the Hillequation. Half-maximal binding occurred at a concentration of 56 pM[³H]resiniferatoxin; the maximal receptor density was 222 fmol per onemillion cells. Note that the Hill coefficient (1.8) is indicative ofpositive binding cooperativity. Three additional experiments yieldedsimilar results. Data are presented as the mean (+/−S.E.M), and bindingparameters are set forth in Example 6, Results.

[0012]FIG. 1B. Specific binding of [³H]resiniferatoxin to HEK 293 cellstransfected stably with a cDNA encoding the rat VR1 (HEK293/VR1 cells).Expanded view of the concentration range from 0-200 pM of the specificbinding of [³H]resiniferatoxin to HEK293/VR1 cells. As a result of thecooperativity index approaching 2, the specific binding curve issigmoidal in this concentration range. Measured values are from FIG. 1A.The binding curve is hypothetical and was computer-generated using thebinding parameters from FIG. 1A.

[0013]FIG. 1C. Specific binding of [³H]resiniferatoxin to HEK 293 cellstransfected stably with a cDNA encoding the rat VR1 (HEK293/VR1 cells).Scatchard plots of specific [³H] resiniferatoxin binding to HEX 293/VR1cells and CHO/VR1 cells. Filled circles indicate data for HEK293/VR1cells; data for CHO/VR1 cells are indicated by triangles. The Scatchardplots are convex due to the positive cooperativity of the binding. Alsonote that the B_(max) value is approximately twice as high in CHO/VR1 asin HEK293/VR1 cells.

[0014]FIG. 2. Scatchard plot of specific [³H] resiniferatoxin binding torat dorsal root ganglion membranes. Half-maximal binding occurred at aconcentration of 60 pM [³H]resiniferatoxin; the maximal receptor densitywas 302 fmol per one million cells. A Hill coefficient, or cooperativityindex, of 1.5 was observed. Compare with FIG. 1C and note that [³H]resiniferatoxin binds to rat dorsal root ganglion membranes expressingnative vanilloid receptors and to mammalian cells (HEK293 and CHO)transfected with the cloned rat vanilloid receptor VR1 with similarbinding parameters. Results of a single experiment are shown; a secondexperiment yielded similar results.

[0015]FIG. 3A. Inhibition of specific [³H] resiniferatoxin binding toHEK 293/VR1 cells by the typical vanilloid agonists olvanil andcapsaicin, the novel vanilloids isovelleral and scutigeral, and thecompetitive vanilloid receptor antagonist capsazepine. Points representvalues from 3 to 6 independent determinations; error bars indicateS.E.M. See Example 6, Results, for calculated K_(I) values.

[0016]FIG. 3B. Inhibition by olvanil, capsaicin, and capsazepine ofspecific [³H] resiniferatoxin binding to HEK293/VR1 cells and to CHO/VR1cells. Open markers indicate data for HEK293/VR1 cells; closed markersindicate data for CHO/VR1 cells. HEK293/VR1 data are from FIG. 3A. ForCHO/VR1 cells, points represent mean values of 2 determinations; errorbars indicate range.

[0017]FIG. 4A. Time dependence of capsaicin and RTX-evoked calciummobilization in CHO/VR1 cells. Time dependence of capsaicin-evokedcalcium mobilization in CHO/VR1 cells in response to 3 nM, 30 nM and3,000 nM capsaicin. In FIG. 4A and FIG. 4b, traces from a representativeside-by-side comparison of capsaicin and resiniferatoxin effects aredisplayed. Note that these two typical vanilloid agonists displaysubstantially different kinetics from the responses they evoke.

[0018]FIG. 4B. Time dependence of resiniferatoxin-evoked calciummobilization in CHO/VR1 cells in response to 0.01 nM, 1 M and 100 nMresiniferatoxin.

DESCRIPTION OF THE SEQUENCE LISTINGS

[0019] SEQ ID NO:1. Human capsaicin receptor cDNA.

[0020] SEQ ID NO:2. Human capsaicin receptor cDNA. Differs from SEQ IDNO:1 only in the 5′ and 3′ untranslated regions.

[0021] SEQ ID NO:3. DNA encoding Short form of Human capsaicin receptor.

[0022] SEQ ID NO:4. Protein encoded by both SEQ ID NO:1 and SEQ ID NO:2.Transmembrane domains span residues thusly: TM1=434-455, TM2=480-495,TM3=510-530, TM4=543-569, TM5=577-596, TM6=656-684.

[0023] SEQ ID NO:5. Protein encoded by SEQ ID NO:3. Transmembranedomains span residues thusly: TM1=434-455, TM2=480-495.

[0024] SEQ ID NO:6. Amino acid sequence of FLAG epitope.

[0025] SEQ ID NO:7. Amino acid sequence of His6x epitope.

[0026] SEQ ID NO:8. Rat capsaicin receptor VR1 cDNA sequence.

[0027] SEQ ID NO:9. Rat capsaicin receptor VR1 amino acid sequence.

[0028] SEQ ID NO:10. The DNA sequence of a 500 bp Bgl II fragment of therat VR1 cDNA (used as a probe).

[0029] SEQ ID NO:11. The DNA sequence of an 830 bp BamHI/NcoI fragmentof the rat VR1 cDNA (used as a probe).

[0030] SEQ ID NO:12. Forward primer used to clone rat VR1 cDNA.

[0031] SEQ ID NO:13. Reverse primer used to clone rat VR1 cDNA.

SUMMARY OF THE INVENTION

[0032] This invention relates to novel human capsaicin receptorpolypeptides and the nucleotide sequences encoding them. Also includedin this invention are nucleic acid vectors (e.g., plasmids) comprisingthe nucleotide sequence encoding these receptors, mammalian andnon-mammalian cell lines comprising such vectors and thereby expressingat least one of the receptor polypeptides, and purified membranesobtained from such mammalian and non-mammalian cell lines. In certainpreferred embodiments the polypeptides are full-length active humancapsaicin receptor proteins that are capable of binding to capsaicin orcapsaicin analogues. In other embodiments the receptors are naturallyoccurring truncated human capsaicin receptor proteins that are expressedat higher levels in bacterial cells than are full-length human capsaicinreceptor proteins. Such truncated proteins are useful for bacterialexpression e.g., for the production of immunogens for use in preparinganti-capsaicin receptor antibodies.

[0033] In another aspect, this invention relates to assays foridentifying compounds that modulate capsaicin receptors, such assaysrequiring recombinantly expressed active human capsaicin receptorproteins that are capable of binding to capsaicin or capsaicinanalogues. Cell lines that express the human capsaicin receptor areuseful for screening compounds for either agonist, reverse agonist orantagonist activity at capsaicin receptors. Compounds that act asagonists at the human capsaicin receptor are useful as flavoring agentsor animal repellents, while those that act as antagonists or reverseagonists may be useful as analgesics or anesthetics.

[0034] In a separate aspect this invention relates to diagnostic assaysfor capsaicin receptor polymorphisms and aberrant capsaicin receptorexpression levels. Such assays are useful for identifying individualsthat are either particularly susceptible or particularly insusceptibleto the types of pain mediated by the capsaicin receptor and thereby fordetermining which individuals will benefit from and which will proverefractory to treatment with modulators of this receptor.

DETAILED DESCRIPTION OF THE INVENTION Nucleic Acids

[0035] The present invention provides isolated nucleic acid moleculesencoding human capsaicin receptors. Isolated nucleic acid moleculesencoding human capsaicin receptors comprise DNA molecules, such asgenomic DNA molecules, cDNA molecules, or RNA molecules. In oneembodiment the isolated nucleic acid molecule is the cDNA sequence shownSEQ ID NO:1. In a separate embodiment the isolated nucleic acid moleculeis the cDNA sequence shown is SEQ ID NO:2. In another embodiment theisolated nucleic acid molecule is the cDNA sequence shown in SEQ IDNO:3. SEQ ID NO:1 and SEQ ID NO:2 differ in their non-coding regions,while SEQ ID NO:3 encodes a shortened (truncated) form of the humancapsaicin receptor, as well as containing a different 5′ non-codingregion.

[0036] The invention also includes an isolated nucleic acid moleculeencoding the amino acid sequence of SEQ ID NO:4 and an isolated nucleicacid molecule encoding the amino acid sequence of SEQ ID NO:5, incertain preferred embodiments the nucleic acid molecules encodepolypeptides with amino acid sequences corresponding to the sequencesstarting with amino acid 2 (Lys) of SEQ ID NO:4 or SEQ ID NO:5. Alsoincluded is an isolated nucleic acid molecule encoding a human capsaicinreceptor sequence comprising the amino acid sequence consistingessentially of SEQ ID NO:5.

[0037] It will be apparent to those skilled in the art that due to thedegeneracy of the genetic code numerous variants of the describednucleic acid molecules can be created by substituting 1 or more codonswithout changing the encoded amino acid sequence of the protein product.Additionally, nucleic acid changes may be made in the non-coding regionof the nucleic acid sequences without altering the amino acid sequenceof the protein product.

[0038] The present invention also encompasses DNA and cDNA sequencesthat encode amino acid sequences which differ from those of the humancapsaicin receptor but which do not produce phenotypic changes.Preferably such changes are conservative amino acid changes. By the term“conservative amino acid change” is meant any change from one amino acidto another amino acid considered to have similar characteristics (see,e.g., Schulz & Schirmer, 1990) as set forth in Table I hereto. TABLE IAmino Acids Grouped by Characteristics Large Basic Side Acidic SideAliphatic Aromatic Polar Side Chains Chains Side Chains Side ChainsChains Lysine, Aspartate, Leucine, Phenylalanine, Glycine, Arginine,Glutamate Isoleucine, Tyrosine, Alanine, Histidine Valine TryptophanProline, Cysteine, Serine, Methionine Threonine, Asparagine, Glutamine

[0039] Also within the scope of the present invention are other changesto DNA and cDNA sequences encoding the amino acid sequences of SEQ IDNO:4 and SEQ ID NO:5 are in-frame additions of nucleic acid sequencesencoding useful amino acid sequence tags. Such tags are useful as, e.g.,antibody recognition sites and as sites contributing strong bindinginteraction characteristics (such as glutathione-S-transferase binding,biotin binding, or metal chelation binding) that are useful forfacilitating protein purification via, e.g. affinity chromatography.Such amino acid sequences are well known in the art, and include, butare not limited to the His-6x epitope (SEQ ID NO:6), which chelatescopper and other metal ions and is specifically bound by the MonoclonalAnti-polyhistidine Clone HIS-1 monoclonal antibody (Sigma, St. LouisNo.H1029), and the FLAG epitope (SEQ ID NO:7), which is specificallybound by the FLAG-M2 monoclonal antibody (Sigma, St. Louis No. F3165).Techniques for making such modifications are also well known in the art,and may be readily carried out using routine methods or by usingcommercially available kits, for example, the Sigma Mammalian FLAGExpression Kits (Sigma, St. Louis, e.g., Nos. FL-MA and FL-MC).

[0040] Also included in the invention are DNA and cDNA sequencesencoding human capsaicin receptors identical to those of SEQ ID NO:4 orSEQ ID NO:5, except in the regions encoding their transmembrane domains(as set forth below in the SEQUENCE LISTING). The transmembrane domainsof the capsaicin receptor are believed to be β strands. Sequencesconsisting essentially of the amino acids Tyrosine, Tryptophan, Valine,Threonine, Glutamine, Methionine, Leucine, Isoleucine, Phenylalanine andCysteine are known to have high propensities for forming β strands (Chouand Fasman, 1974). Substitution of nucleotides encoding any of theseamino acids for nucleotides encoding other amino acids in thetransmembrane regions of the capsaicin receptors of SEQ ID NO:4 and SEQID NO:5 will result in functional receptor translation products, and arewithin the scope of the present invention.

Polypeptides

[0041] This invention provides isolated human capsaicin receptorpolypeptides having the amino acid sequence of SEQ ID NO:4 or SEQ IDNO:5. In certain preferred embodiments the polypeptides have amino acidsequences corresponding to the sequences starting with amino acid 2(Lys) of SEQ ID NO:4 or SEQ ID NO:5. The amino acid sequence given bySEQ ID NO:4 is the protein product encoded by both SEQ ID NO:1 and SEQID NO:2. The amino acid sequence given by SEQ ID NO:5 is the proteinproduct encoded by SEQ ID NO:3.

[0042] This invention also encompasses human capsaicin receptors havingamino acid sequences that further differ from those exemplified herein,but which do not exhibit phenotypic changes. Such amino acid sequencesare described above in the discussion of nucleotide sequences, andfurther include human capsaicin receptors having amino acid sequencesthat differ from those of SEQ ID NO:4 and SEQ ID NO:5 in thetransmembrane domains of the protein without eliminating capsaicinreceptor binding or signaling functions. The regions of the amino acidsequences for the receptor considered to represent the transmembranedomains of the protein are annotated as TM1, TM2, TM3, TM4, TM5 and TM6in the sequence listing for SEQ ID NO:4 and SEQ ID NO:5. It is predictedthat the transmembrane domains of the capsaicin receptor are β strandsThe amino acids Tyrosine, Tryptophan, Valine, Threonine, Glutamine,Methionine, Leucine, Isoleucine, Phenylalanine and Cysteine are known tohave high propensities for being in β strands (Chou and Fasman, 1974).Amino acid sequences of the human capsaicin receptor having of any ofthese amino acids substituted for other amino acids in the transmembranedomains are encompassed by this invention.

Vectors Encoding the Human Capsaicin Receptor

[0043] The present invention additionally provides nucleic acid vectorscomprising a nucleic acid sequence encoding a polypeptide with the aminoacid sequence of SEQ ID NO:4 or SEQ ID NO:5 or, in certain preferredembodiments, encoding amino acid sequences corresponding to thesequences starting with amino acid 2 (Lys) of SEQ ID NO:4 or SEQ IDNO:5. Such nucleic acid sequences include any of the above-describednucleic acid sequences. Suitable vectors include, but are not limitedto, a plasmid or a viral vectors. In order for the vector to be used forrecombinant expression of a capsaicin receptor, the nucleic acidsequence encoding the receptor must be operatively linked to a nucleicacid sequence comprising at least one regulatory element in theappropriate orientation for expression. Such regulatory elements arewell known to those of skill in the art. Preferred regulatory elementsare heterologous regulatory elements, i.e., regulatory elements that arenot naturally found operatively linked to nucleic acid sequencesencoding human capsaicin receptor polypeptides. Such elements includethose obtained from other mammalian species as well as those fromnon-mammalian vertebrates, invertebrates, microbes and viruses.Particualrly preferered regulatory elements are inducible elements,i.e., elements that do not always stimulate expression (or onlystimulate relatively low levels of expression), but that respond toenvironmental stimuli by increasing expression levels of the operativelylinked coding sequences. A preferred inducible element is thetetracycline repressible element found in the commercially availablepTET OFF™ plasmid vector.

[0044] Propagation of the vectors of the invention in microbial hosts isfacilitated by the presence in the vector of sequences that act asorigins of replication for microbial DNA synthesis, but suchmicrobial-specific sequences are considered to reduce the rate ofsuccess in generating certain recombinant cells (particularly ingenerating transgenic animals), and thus such microbial sequences may bebeneficially excised (e.g., by restriction enzyme digestion) prior tothe introduction of the vector into an animal cell (e.g., a vertebratezygote).

[0045] The vectors of the invention may be transformed, transfected,microinjected, or otherwise introduced into suitable host cells to formhost cell-vector systems for the expression of a polypeptide of theinvention. In certain preferred embodiments, the polypeptide exhibitshuman capsaicin receptor binding and/or signaling activity.

[0046] This invention encompasses any of the above-described vectorsadapted for infection or transformation of a bacterial cell. Bacterialexpression systems for the expression of membrane receptor proteins areavailable (Muench, et al. 1995). Bacterial host vector systems are alsouseful in that (when the appropriate microbial origin of DNA replicationis present in the vector) they allow the production of large quantitiesof DNA or (when an appropriate microbial RNA polymerase transcriptioninitiation site is present in the appropriate orientation for RNAexpression) of RNA encoding the polypeptides of the invention. Aparticularly preferred vector for the bacterial expression of thepolypeptides of SEQ ID NO:5 is the be pRSET vector that is commerciallyavailable from Invitrogen (Carlsbad, Calif.). Protein expression usingthis vector can be conveniently induced by adding the lactose analogisopropylthiogalactoside (IPTG) to the bacterial culture medium.

[0047] This invention also encompasses the above-described vectoradapted for expression in a eucaryotic cell (preferably an insect cell,an amphibian cell, or a mammalian cell) which vector further comprisesheterologous regulatory elements allowing expression in the celloperatively linked to a nucleic acid molecule encoding at least onepolypeptide of the invention, so as to permit expression thereof.

[0048] In one embodiment, the vector is adapted for expression in aninsect cell. Plasmids commonly used to generate such vectors for thispurpose include the BacPak8 and BacPak9 baculoviral vector plasmids(Clontech, Palo Alto, Calif.). These plasmid vectors typically include amultiple cloning site for the insertion in the appropriate orientationfor expression of a DNA fragment comprising the sequence encoding thepolypeptide to be expressed, such as any of the cDNA sequences of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3, a promoter sequence, a bacterialorigin of DNA replication, and markers for antibiotic resistance.

[0049] This invention also encompasses the above-described vectoradapted for expression in an amphibian cell, preferably a Xenopus laevisoocyte. Plasmids that may be used to generate such vectors for thispurpose include the pcDNA3.1 vector (Invitrogen, Carlsbad, Calif.).

[0050] In a preferred embodiment, the vector is adapted for expressionin a mammalian cell. An example of a plasmid commonly used to generatesuch vectors for expression of polypeptides (e.g., those of the presentinvention) in mammalian cells is pBK-CMV (Stratagene, La Jolla, Calif.)in which the regulatory elements include the cytomegalovirus promoter,which is activated by proteins that are ubiquitously expressed invertebrate cells.

[0051] This invention provides plasmid vectors designated PT35, PT36,and PT44, which comprise the regulatory elements necessary forexpression of DNA in a mammalian cell operatively linked to the DNAencoding human capsaicin receptor polypeptides of the invention in theappropriate orientation so as to permit expression.

[0052] These plasmids, PT35, PT36, and PT44 were deposited on Aug. 27,1999 with the American Type Culture Collection (ATCC) 10801 UniversityBlvd., Manassas, Va. 20110-2209, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and were accordedPatent Deposit Numbers PTA-576, PTA-577 and PTA-578, respectively. Inaccordance with 37 CFR 1.808 (a)(2), et seq., all restrictions imposedby the depositor on the availability to the public of the depositedmaterials will be irrevocably removed upon the granting of a U.S. patentfrom the present application.

Recombinant Cells Expressing the Human Capsaicin Receptor

[0053] In another aspect this invention provides a recombinant cellexpressing the human capsaicin receptor polypeptides, said cell havingbeen obtained by adding the above-described vectors to a host cell. Suchhost cells include cells from cell lines, cells from primary cultures,and ova and oocytes A preferred cell of this type is a Xenopus laevisoocyte recombinantly expressing active human capsaicin receptor proteinsof the invention. Other preferred recombinant cells of the inventioninclude other amphibian cells, insect cells, or mammalian cellscomprising at least one of the above-described vectors of the invention.The present invention thus includes such cells comprising such vectorscomprising at least the coding regions of the nucleic acid sequences ofat least one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, operativelylinked to regulatory sequences in the appropriate orientation for theexpression of the encoded polypeptides of the invention.

[0054] The vector may be added to the host cell by any of the meanscommonly practiced by those skilled in the art, including but notlimited to infection, transformation, transfection or microinjection.Resulting recombinant cells (typically the progeny of thevector-containing host cell) will preferably express at least 2-foldgreater capsaicin or capsaicin agonist binding activity (measured, e.g.,as described for vanilloid binding in Example 6 hereto), or morepreferably at least 10-fold greater binding activity, or most preferablyat least 20-fold greater binding activity than a control host cell towhich vector has not been added.

[0055] In one embodiment the recombinant cell is an insect cellcomprising the above-described plasmid or vector. One commonly usedinsect cell protein expression system is that of Spodoptera frugiperdacells infected with a baculovirus vector.

[0056] The invention further provides a mammalian cell comprising theabove-described vector. Both adherent and non-adherent mammalian cellare appropriate for expression of the human capsaicin receptorpolypeptides of the invention. The mammalian cell may be a COS-7 cell,CHO cell, human embryonic kidney 293 cell (HEK 293), a U937 cell or anyother suitable mammalian cell, including cells in primary cell cultures.The mammalian cell may also be prepared by generating a transgenicanimal (preferably the animal is a rodent or a domestic livestockspecies such as a pig, cow, got, sheep, rabbit, or chicken). Methods forgenerating such transgenic animals are well known, and while often oflow efficiency and technically demanding, are nonetheless routine in theart.

[0057] Artificially high expression levels of ion channels typicallyhave toxic effects on cultured cells. Without wishing to be bound by anyparticular theory of operation, it is believed that when it occurs, suchtoxicity results from excess entry of ions (e.g., Ca²⁺) into cellsexpressing artificially high levels of ion channels. The capsaicinreceptors of the invention act as ion channels, and their expression atartificially high levels would thus be expected to be limited by suchtoxicity.

[0058] The present invention provides for recombinant mammalian cells(e.g., cells comprising an expression vector) expressing high levels ofcapsaicin receptors, or capable of expressing high levels of capsaicinreceptors (e.g., upon derepression—for example removal of tetracyclinefrom the growth medium of cells comprising expression vectors of theinvention using the regulatory elements of the commercial pTET OFF™inducible plasmid vector). Such cells express higher numbers ofcapsaicin binding sites than do naturally occurring cells, and areparticularly useful for the preparation of membranes containingcapsaicin receptors for use in binding assays for the identification ofcompounds that interact (preferably ones that specifically interact,e.g., by binding to the ligand-binding site of the receptor) with suchreceptors. Preferably, the recombinant cells of the invention are stablytransfected cells.

[0059] Preferred cells express at least 5×10⁴ capsaicin receptor ligandbinding sites per cell. Preferably the cells express at least 1.5×10⁵such binding sites per cell. More preferably the cells express at least3×10⁵, and even more preferably, 3×10⁶ such binding sites per cell. Mostpreferably, the cells express at least 10⁷ capsaicin receptor ligandbinding sites per cell.

[0060] Preferred cells express at least 80 fmol of capsaicin receptormolecules per 10⁶ cells. Preferably the cells express at least 250 fmolof capsaicin receptor molecules per 10⁶ cells. More preferably the cellsexpress at least 470 fmol of capsaicin receptor molecules per 10⁶ cellsand even more preferably at least 2 pmol of capsaicin receptor moleculesper 10⁶ cells. Most preferably, the cells express at least 16 pmol ofcapsaicin receptor molecules per 10⁶ cells.

Isolated Membranes of Recombinant Cells

[0061] In certain of its aspects the present invention providespreparations comprising isolated membranes of the recombinant cells ofthe invention. Preferably, the isolated membranes should exhibitcapsaicin receptor ligand binding activity that is significantlygreater, preferably at least 2-fold greater, more preferably at least10-fold greater and most preferably at least 20-fold greater than thatexhibited by control membranes isolated from a control host cell (e.g.,a cell of the same cell line used to prepare the recombinant cell of theinvention that does not contain any vector, or contains a control vectorthat does not encode a capsaicin receptor). Preferred membranes contain,per mg of total membrane protein, at least 415 fmol, preferably at least1.25 pmol, even more preferably 2.35 pmol, particularly preferably 4.2pmol, and most preferably at least 25 pmol of capsaicin receptor.Membranes can be isolated by any suitable method, such as any of themembrane preparation methods that are routinely used in the art.

Assays for Identifying Modulators of Capsaicin Receptors

[0062] In a final aspect, the invention provides methods for determiningwhether a compound can specifically bind to a capsaicin receptor andmethods for determining whether a compound can modulate a capsaicinreceptor as either an agonist or an antagonist. Agonist compounds areuseful as analgesics. This counter-intuitive result is believed tofollow from prolonged receptor desensitization that can occur followingexposure to such compounds. Agonists, antagonists and reverse agonistsare all useful as analgesics, as well as for the prevention andtreatment of other conditions, such as treatment of urinaryincontinence, prevention of urinary bladder hyper-reflexia and treatmentof certain neuropathic pain states such as post herpetic neuralgia,diabetic neuropathy, carpal tunnel syndrome and phantom limb pain inamputees.

[0063] The invention thus provides assays for identifying compoundsuseful a) as analgesics, b) for the treatment of urinary incontinence,c) for the prevention of urinary bladder hyper-reflexia and d) for thetreatment of neuropathic pain states (e.g., post herpetic neuralgia,diabetic neuropathy, carpal tunnel syndrome or phantom limb pain).

[0064] The invention in particular provides an assay for determining ifa compound binds specifically to capsaicin receptors. This assaycomprises contacting an experimental sample of either recombinant cellsof the invention or isolated membrane preparation of such cells with alabeled capsaicin agonist and a test compound. A second control sampleof either recombinant cells expressing the human capsaicin receptor oran isolated membrane preparation of such cells is contacted only withlabeled capsaicin agonist. The unbound labeled agonist is removed fromboth samples and the amount of bound label in both the experimentalsample and the control sample is determined. The amount of bound labelin the experimental sample is compared to the amount bound label in thecontrol sample. If the experimental sample exhibits a 2-fold decrease,or more preferably a 5-fold decrease or most preferably a 10-folddecrease in the amount of bound labeled capsaicin agonist the compoundin the experimental sample is identified as binding specifically tocapsaicin receptors.

[0065] In the above-described binding assay the labeled capsaicinagonist may be any agonist that is known to bind specifically tocapsaicin receptors, such as capsaicin or resiniferatoxin and may belabeled by any detectable label. Detectable labels include, but are notlimited to, radiolabels, fluorescent labels and colorometric labels. Aparticularly preferred labeled capsaicin agonist is [³H]resiniferatoxin. Removal of unbound label may be accomplished byfiltering or washing the samples but is not limited to these methods.

[0066] The invention also provides functional assays for identifyingcompounds that act as modulators of capsaicin receptors. Such assays canbe used to classify compounds as agonists or antagonists of thecapsaicin receptor.

[0067] This invention provides a method for determining whether acompound is a human capsaicin receptor agonist, which comprisescontacting a recombinant cell of the invention with the compound underconditions that permit activation of a functional human capsaicinreceptor response, detecting a functional increase in human capsaicinreceptor activity, and there by determining whether the compound is ahuman capsaicin receptor agonist.

[0068] In one such embodiment the invention provides an assay fordetermining if a compound is an agonist of capsaicin receptors where thefunctional response is a change in the concentration of intracellularCa²⁺. This assay comprises contacting a sample of recombinant cellsexpressing the human capsaicin receptor with an indicator ofintracellular Ca²⁺ concentration to yield indicator-loaded cells. Aftera sufficient incubation period excess indicator is removed from thecells to yield washed, indicator-loaded cells. A potential agonistcompound is added to a sample of the washed, indicator-loaded cells.This sample is the experimental sample; the control sample is comprisedof washed, indicator-loaded cells to which no potential agonist compoundhad been added. The concentrations of intracellular Ca²⁺ experimentaland control samples are measured by quantitating a change in theindicator of intracellular Ca²⁺. The concentration of intracellular Ca²⁺in the experimental cells that have been contacted with a potentialagonist compound is compared to the concentration of intracellular Ca²⁺in the control cells. If the experimental sample exhibits a 1.5-foldincrease, or more preferably a 5-fold increase or most preferably a10-fold increase (or any significant increase) in the concentration ofintracellular Ca²⁺ the compound in the experimental sample is identifiedas a capsaicin receptor agonist. As used herein and in the claims, asignificant change (e.g., increse or decrese) is one that is significantto the p≦0.05 level in any standard parametric test of statisticalsignificance, such as the F-test, or the Student's T-test.

[0069] Particularly preferred indicators of intracellular Ca²⁺concentration are membrane permeable calcium sensitive dyes, e.g.,Fluo-3 and Fura-2. These dyes produce a fluorescent signal when bound toCa²⁺. Removal of excess indicator from the indicator-loaded cells may beaccomplished by washing or filtering cells, but is not limited to thesemethods.

[0070] This invention provides a method for determining whether acompound is a human capsaicin receptor antagonist, which comprisescontacting a cell of the invention with the compound in the presence ofa known capsaicin receptor agonist, such as capsaicin orresiniferatoxin, under conditions that permit the activation of afunctional capsaicin receptor response, detecting a decrease in humancapsaicin receptor activity, and thereby determining whether thecompound is a human capsaicin receptor antagonist.

[0071] In one embodiment, the assay to identify compounds that act asantagonists of capsaicin receptors comprises contacting a test sample ofrecombinant cells expressing the human capsaicin receptor with anindicator of intracellular Ca²⁺ concentration and a test compound(prefereably the cells are pre-loaded with the indicator). A secondcontrol sample of recombinant cells expressing the human capsaicinreceptor is contacted only with the indicator of intracellular Ca²⁺concentration. After a sufficient incubation period excess indicator ofintracellular Ca²⁺ is removed from the test and control cells to yieldwashed, indicator-loaded test and control cells. An agonist of thecapsaicin receptor is added to the washed, indicator-loaded cells toyield agonist-contacted test cells and agonist-contacted control cells.The concentration of intracellular Ca²⁺ in the agonist-contacted testcells and the agonist-contacted control cells is measured by measuringchanges in the properties of the indicator of intracellular Ca²⁺concentration. The concentration of intracellular Ca²⁺ in theagonist-contacted test cells is compared to that in agonist-contactedcontrol cells. A test compound for which this comparison indicates thatthe concentration of intracellular Ca²⁺ in the agonist-contacted testcells is significantly less, to the p≦0.05 level, than the concentrationof intracellular Ca²⁺ in the agonist-contacted control cells isidentified as an antagonist of capsaicin receptors.

[0072] As in the assay for agonists of the capsaicin receptor,particularly preferred indicators of intracellular Ca²⁺ concentrationare the membrane permeable calcium sensitive dyes, Fluo-3 and Fura-2.These dyes produce a fluorescent signal when bound to Ca²⁺. Removal ofexcess indicator from the indicator-loaded cells may be accomplished byany suitable method, such as washing or filtering cells.

[0073] The invention will now be further described with reference to thefollowing examples.

EXAMPLES Example 1 Isolation of Human Capsaicin Receptor DNA Clones

[0074] Poly A+ RNA was isolated from frozen human dorsal root ganglia. Acomplementary DNA (cDNA) library was constructed using the ZAP EXPRESScDNA SYNTHESIS KIT and the ZAP EXPRESS cDNA GIGAPAK III GOLD CLONING KIT(Stratagene, La Jolla, Calif.) according to the manufacturer'sinstructions. This library contained approximately 1.1×10⁶ independentclones. 1×10⁶ plaque forming units (PFU) from this library were platedand transferred to nitrocellulose filters for hybridization screening.Two ³²P labeled probes were used for screening the filters. One probe,SEQ ID NO:10, corresponded to a 500 bp Bgl II fragment of the publishedrat Vanilloid Receptor 1 cDNA (VR1; Caterina, et al. 1997) The secondprobe, SEQ ID NO:11, corresponded to an 830 bp BamHI/Nco I fragment ofVR1. Filters were hybridized with either probe overnight at 42° C. andsubsequently washed at 55° C. in 0.2× SSC/0.1% SDS. Positive phage wereisolated and re-screened to obtain a single (clonal) phage. The pBK-CMVphagemid was excised from the clonal lambda phage by using the EXASSISTHelper phage (Stratagene). The resulting plasmids were transformed intoE. coli strain XLOLR (Stratagene). Plasmid DNA was isolated by maxi prepfor sequencing. The nucleotide sequences of the EcoRI/XhoI inserts ofthe phagemid clones were determined with an ABI CYCLE SEQUENCING KIT(Perkin-Elmer Applied Biosystems, Foster City, Calif.).

Example 2 Recombinant Cells

[0075] A. Transiently Transfected Cells

[0076] Plasmids PT35, PT36 and PT44 were isolated from XLOLR E. coli andseparately transfected into HEK 293T cells (Edge Biosystems,Gaithersville, Md.) by adding 2 μg plasmid DNA and 12 μl LIPOFECTAMINEReagent (Life Technologies cat no. 10964-013, Life Technologies,Gaithersville, Md.) per 60 mm culture plate according to themanufacturer's instructions. Cells were grown for 24 hrs at 37° C. thenreseeded onto 96-well plates suitable for use in the FLIPR™ Plate Reader(Molecular Devices, Sunnyvale Calif.). Cells were grown an additional24-48 hours before assay.

[0077] Transfected cells were assayed for changes in detectible Ca²⁺levels using the Calcium mobilization assay described below in Example5. The results of these experiments are set forth in Table II.

[0078] B. Stably Transfected Calls

[0079] Cells stably expressing the human capsaicin receptor are selectedusing the Neomycin resistance marker present in the PT35, PT36 and PT44plasmids. HEK 293T cells transiently transfected with the PT35, PT36 orPT44 plasmids as described above are grown in media containing theantibiotic G418 for two weeks to isolate cell lines stably expressingthe recombinantly expressed human capsaicin receptor.

[0080] C. Inducible Stably Transfected Cells

[0081] The cDNA encoding SEQ ID NO:4 is subcloned into the pTRE vector(Clontech, Palo Alto, Calif.) for recombinant expression in mammaliancells. Plasmids are transfected with LIPOFECTAMINE™ into Chinese HamsterOvary (CHO) cells containing the pTET OFF™ Regulator plasmid (Clontech,Palo Alto, Calif.). In these cells, expression of the pTRE plasmid isrepressed in the presence of tetracycline but is induced upon removal ofthe antibiotic. Stable clones are isolated in culture medium containingpuromycin (10 μg/ml) and maintained in medium supplemented withtetracycline (1 μg/ml). Cells are grown without antibiotic for 48-72hours prior to assay to facilitate maximal expression of the humancapsaicin receptor.

Example 3 Purified Membranes of Cells Expressing the Human CapsaicinReceptor

[0082] The human capsaicin receptor-transfected HEK 293T cells ofExample 2 were seeded into 96 well plates and grown to 70-90%confluency. Cells were harvested by centrifugation at 3500× g and washedonce by resuspension in ice cold PBS containing protease inhibitors.Cells were lysed by POLYTRON (speed 5, 30 seconds) and centrifuged at536× g for 10 minutes in order to remove DNA, cellular organelles, andunlysed cells. The supernatant, containing isolated membranes, wasdecanted to a clean centrifuge tube and centrifuged for 20 minutes at40,000× g. The resulting pellet was washed twice in ice cold PBS andcentrifuged again for 20 minutes at 40,000× g. The supernatant of thisstep was discarded. The protein concentration of the resulting membranepellet was measured using the Bio-Rad (Bradford) protein assay. By thismeasure, a 1 liter culture of cells typically yielded 50-75 mg of totalmembrane protein.

Example 4 Radioligand Binding Assay for Modulators of CapsaicinReceptors

[0083] Binding studies with [³H] resiniferatoxin (RTX) (NEN, Boston)were carried out according to the protocol of Szallasi, et. al (1992) inwhich non-specific RTX Binding is reduced by adding bovineα₁-glycoprotein (100 μg per tube) after the binding reaction has beenterminated. Binding assay mixtures were set up on ice and contained [³H]RTX, non-radioactive ligands, 0.25 mg/ml bovine serum albumin (Cohnfraction V), and 5×10⁴-1×10⁵ human capsaicin receptor-transfectedHEK-293T cells. The final volume was adjusted to 500 μl (competitionbinding assays) or 1.000 μl (saturation binding assays) with the bufferdescribed above. Non-specific binding was defined as that occurring inthe presence of 1 μM non-radioactive RTX. For saturation binding, [³H]RTX was added in the concentration range of 7-1,000 pM, using 1 to 2dilutions. Competition binding assays were performed in the presence of60 pM [³H] RTX and various concentrations of competing ligands. Thebinding reaction was initiated by transferring the assay mixtures into a37° C. water bath and was terminated following a 60 minute incubationperiod by cooling the tubes on ice. Membrane-bound RTX was separatedfrom free RTX as well as the α₁-glycoprotein-bound RTX by pelleting themembranes in a Beckman 12 benchtop centrifuge for 15 minutes at 14,000×g. The radioactivity of membrane-bound RTX was determined byscintillation counting. Equilibrium binding parameters were determinedby fitting the Hill equation to the measured values (Szallasi, et al.,1993) with the aid of the computer program FitP™ (Biosoft, Ferguson,Mo.).

Example 5 Calcium Mobilization Assays

[0084] A. Response to Capsaicin or Resiniferatoxin

[0085] Human capsaicin receptor transfected HEK 293T cells were seededinto 96 well plates and grown to 70-90% confluency. The cells were thenwashed once with Krebs Ringer solution. Fluo-3 (Molecular Probes,Eugene, Oreg.) calcium sensitive dye (10 ug/mL) was added and incubatedwith the cells at room temperature for 1 to 2 hours. The 96 well plateswere then washed to remove excess dye. Fluorescence response wasmonitored upon the addition of either 300 nM capsaicin or 30 nMresiniferatoxin by a FLIPR™ plate reader (Molecular Devices, SunnyvaleCalif.) by excitation an 480 nM and emission at 530 nm. Cellstransfected with plasmids PT35 and PT44, encoding the full length humancapsaicin receptor (SEQ ID NO:1 and SEQ ID NO:2), typically exhibitedsignals of 5,000-8,000 Arbitrary Fluorescent Light Units in response toagonist. TABLE II Cell Type Ave Max Response (RFU) Wild-Type  843 PT357257 PT36  805 PT44 5529

[0086] B. Assays for the Identification of Receptor Agonists andAntagonists

[0087] The calcium mobilization assay described above may be adapted foridentifying test compounds as having agonist or antagonist activity atthe human capsaicin receptor.

[0088] In order to identify agonist compounds, recombinant cells of theinvention are washed and incubated with Fluo-3 dye as described above. Asubset of the incubated cells are then exposed to a 1 μm concentrationof at least one candidate agonist compound and the fluorescence responseis monitored using a FLIPR™ plate reader (Molecular Devices, Sunnyvale,Calif.). Agonist compounds elicit a fluorescence response at least2-fold that of recombinant cells exposed only to Fluo-3 dye. Preferredagonists elicit a fluorescence response at least 10 fold, and morepreferred agonists elicit a fluorescence response at least 20-fold thatof recombinant cells exposed only to Fluo-3 dye.

[0089] In order to identify antagonist compounds, recombinant cells ofthe invention are washed an incubated with Fluo-3 dye as describedabove. One hour prior to measuring the fluorescence signal, a subset ofthe cells is incubated with a 1 μm concentration of at least onecandidate antagonist compound. The fluorescence response upon thesubsequent addition of either 300 nM capsaicin or 10 nM resiniferatoxinis monitored using a FLIP™ plate reader (Molecular Devices). Agonistcompounds elicit at least a 2-fold decrease in the fluorescence responserelative to that measured in the presence of capsaicin or RTX alone.Preferred antagonist compounds elicit at least a 10 fold, and morepreferred antagonists at least a 20-fold decrease in the fluorescenceresponse relative to that measured in the presence of capsaicin or RTXalone.

Example 6 Characterization of Capsaicin Receptors Expressed at HighLevels in Cultured Cells

[0090] [³H]Resiniferatoxin (RTX) binding and calcium uptake by ratdorsal root ganglion (DRG) neurons show distinct structure-activityrelations, suggestive of independent vanilloid receptor (VR) subtypes.To evaluate the hypothesis that binding and calcium uptake detect twodistinct classes of VRs in DRG neurons, characterization of RTX bindingto the rat capsaicin receptor, VR1, expressed in HEK293 and CHO cellsand comparison of the structure-activity relations with those forcalcium mobilization was tested. In these binding experiments bothtypical (capsaicin and olvanil) and novel (isovelleral and scutigeral)vanilloids were included, as well as the competitive VR antagonistcapsazepine. Vanilloid binding to HEK293/VR1 cells was compared to thatmeasured in rat DRG neurons expressing native VRs. Calcium mobilizationin HEK293/VR1 or CHO/VR1 cells was determined in response to RTX,olvanil, and capsaicin, using a fluorescent method. In addition,capsaicin-induced calcium mobilization in the VR1-transfected cells wasmeasured in the presence of capsazepine or the so-called functional VRantagonist, ruthenium red. Agonist and antagonist potencies determinedin the calcium mobilization assays using HEK293/VR1 or CHO/VR1 cellswere compared to values measured previously in this laboratory forvanilloid-induced ⁴⁵Ca²⁺-uptake by intact rat DRG neurons.

[0091] HEK293/VR1 cells and CHO/VR1 cells bound [³H]RTX with affinitiesof 84 pM and 103 pM, respectively, with a positive cooperativity (Hillnumbers were 2.1 and 1.8). These binding parameters are similar to thosedetermined using rat DRG membranes expressing native rat VRs (a K_(d) of70 pM and a Hill number of 1.7). The typical vanilloid agonists olvaniland capsaicin inhibited [³H]RTX binding to HEK293/VR1 cells with K_(i)values of 0.4 μM and 4.0 μM, respectively. The corresponding values inDRG membranes were 0.3 μM and 2.5 μM. HEK293/VR1 cells and DRG membranesalso recognized the novel vanilloids isovelleral and scutigeral withsimilar affinities (18 and 20 uM in HEK293/VR1 cells; 24 and 21 μM inDRGs). The competitive vanilloid receptor antagonist capsazepineinhibited [³H]RTX binding to HEK293/VR1 cells with a K_(i) value of 6.2μM, and to DRG membranes with an affinity of 8.6 μM. RTX and capsaicininduced calcium mobilization in HEK293/VR1 cells with EC₅₀ values of 4.1nM and 82 nM, respectively. Thus, the relative potencies of RTX (morepotent for binding) and capsaicin (more potent for calcium mobilization)are similar in DRG neurons and cells transfected with VR1. We concludethat VR1 may account for both the ligand binding and calcium uptakeobserved in rat DRG neurons.

[0092] Experimental Procedures

[0093] Materials. [³H]Resiniferatoxin (RTX; 37 Ci/mmol) was synthesizedby the Chemical Synthesis and Analysis Laboratory, NCI-FCRDC, Frederick,Md. Nonradioactive RTX was purchased from Alexis Corp. (San Diego,Calif.) and capsazepine was from RBI (Natick, Mass.). Olvanil was agenerous gift from Procter and Gamble Corp (Cincinnati, Ohio).Isovelleral and scutigeral were donated by Olov Sterner (Lund Univ.,Sweden). All the other chemicals used were purchased from Sigma (St.Louis, Mo.) unless indicated otherwise.

[0094] Molecular Biology. A cDNA encoding the rat vanilloid receptor VR1was cloned from rat dorsal root ganglion (DRG) total RNA by reversetranscription-polymerase chain reaction using primers based on thepublished nucleotide sequence (Caterina et al., 1997). The forwardprimer is set forth below as SEQ ID NO:12 and the reverse primer is setforth below as SEQ ID NO:13. A 2.7 kb cDNA was isolated and thenucleotide sequence was verified to be identical to the publishedsequence, SEQ ID NO:8. This cDNA was subcloned into pcDNA3.1(Invitrogen, Carlsbad, Calif.) and pTRE (Clontech, Palo Alto, Calif.)for recombinant expression in mammalian cells of the encoded VR1polypeptide, SEQ ID NO:9.

[0095] Cell Culture. The pcDNA3.1 VR1 plasmid was transfected into humanembryonic kidney (HEK293) cells using standard methods. Thesetransfected cells were selected for two weeks in media containing G418(400 μg/ml) and then maintained as a pool of stably transfected cells.The pTRE VR1 plasmid was transfected into Chinese Hamster Ovary (CHO)cells containing the pTet Off Regulator plasmid (Clontech). In thesecells, expression of the pTRE plasmid is repressed in the presence oftetracycline but is induced upon removal of the antibiotic. Stableclones were isolated in culture medium containing puromvcin 10 (μg/ml)and maintained in medium supplemented with tetracycline (1 μg/ml) Cellsutilized for assays were grown in culture medium without antibiotic for48-72 hours prior to use. For radioligand binding experiments, cellswere seeded in T175 cell culture flasks in media without antibiotics andgrown to approximately 90% confluency. The flasks were then washed withPBS and harvested in PBS containing 5 mM EDTA. The cells were pelletedby gentle centrifugation and stored at −80° C. until assayed. Forcalcium mobilization assays, cells were seeded into 96-well plates andgrown to 70-90% confluency.

[0096] Membrane Preparations. Female Sprague-Dawley rats weighing200-250 g were euthanized under CO₂ anesthesia. The spinal columns wereopened and DRGs were collected from all levels into ice-coldphysiological saline. DRGs were disrupted with the aid of a tissuehomogenizer in an ice-cold buffer (pH 7.4) containing (in mM) KCl 5,NaCl 5.8, CaCl₂ 0.75, MgCl₂ 2, sucrose 320, and HEPES 10. Tissuehomogenates were first centrifuged for 10 min at 1000× g (4° C.) toremove the nuclear fraction and debris and then the supernatant from thefirst centrifugation was further centrifuged for 30 min at 35,000× g (4°C.) to obtain a partially purified membrane fraction. Membranesresuspended in the homogenization buffer were stored at −80° C. untilassayed.

[0097] Radioligand Binding. Binding studies with [³H]RTX were carriedout according to a published protocol (Szallasi et al., 1992) in whichnon-specific RTX binding is reduced by adding bovine alpha₁ acidglycoprotein (100 μg per tube) after the binding reaction has beenterminated. Binding assay mixtures were set up on ice and contained[³H]RTX, non-radioactive ligands, 0.25 mg/ml bovine serum albumin (Cohnfraction V), and either 5×10⁴-1×10⁻⁵ VR1-transfected cells or isolatedDRG membranes corresponding to 40 μg of DRG membrane protein. The finalvolume was adjusted to 500 μl (competition binding assays) or 1,000 μl(saturation binding assays) with the ice-cold HEPES (pH 7.4) buffersolution described above. Non-specific binding was defined as thatoccurring in the presence of 1 μM non-radioactive RTX. For saturationbinding, [³H]RTX was added in the concentration range of 7-1,000 pM,using 1 to 2 dilutions. Competition binding assays were performed in thepresence of 30 pM (for DRG membranes) or 60 pM (for VR1-transfectedcells) [³H]RTX and various concentrations of competing ligands. Thebinding reactions were initiated by transferring the assay mixtures intoa 37° C. water bath and were terminated following a 60 min incubationperiod by cooling the tubes on ice. Membrane-bound RTX was separatedfrom free, as well as any alpha₁-acid glycoprotein-bound RTX, bypelleting the membranes in a Beckman 12 benchtop centrifuge (15 min,maximal velocity) and the radioactivity determined by scintillationcounting. Equilibrium binding parameters were determined by fitting theallosteric Hill equation to the measured values with the aid of thecomputer program FitP™ (Biosoft, Ferguson, Mo.) as described previously(Szallasi et al., 1993).

[0098] Calcium Mobilization Assays. VR1-transfected cells were seededinto 96-well plates and grown to 70-90% confluency. The cells were thenwashed once with Krebs-Ringer HEPES buffer (25 mM HEPES, 5 mM KCl, 0.96mM NaH₂PO₄, 1 IM MgSO₄, 2 mM CaCl₂, 5 mM glucose, 1 mM probenecid, pH7.4) and resuspended and incubated for 1-2 hours in the above buffersupplemented with FLUO3-AM (2.5-10 μg/ml; Teflabs, Austin, Tex. orMolecular Probes, Eugene, Oreg.) at 37° C. in an environment containing5% CO₂. In some experiments (as indicated below in the RESULTS), theKrebs-Ringer HEPES buffer was also supplemented with 1 mg/ml bovineserum albumin (Cohn fraction V). The wells were then washed twice withKrebs Ringer HEPES buffer. Agonist (olvanil, capsaicin, or RTX)-inducedcalcium mobilization was monitored using either FLUOROSKAN ASCENT(Labsystems, Franklin, Mass.) or FLIPR (Molecular Devices, Sunnyvale,Calif.) instruments. Similarly, varying concentrations of theantagonists ruthenium red or capsazepine were added to cellsconcurrently with agonist (25-50 nM capsaicin). Fluorescence data werecollected to 60-180 seconds and the maximum fluorescence signal wasdetermined. For the capsaicin- and olvanil-induced calcium responses,data obtained between 30 and 60 seconds after agonist application wereused to generate the EC₅₀ values. Kaleidagraph software (SynergySoftware, Reading, Pa.) was utilized to fit the data to the equation:

y=a*(1/(1+(b/x)^(c)))

[0099] to determine the EC₅₀ for the response. In this equation, y isthe maximum fluorescence signal, x is the concentration of the agonistor antagonist, a is the E_(max), b corresponds to the EC₅₀ or IC₅₀value, and finaly, c is the Hill coefficient.

[0100] Results

[0101] Rat Capsaicin Receptor VR1-Transfected Mammalian Cells (HEK293and CHO) and Rat DRG Membranes Expressing Native Rat Vanilloid ReceptorsBind [³H]RTX with Similar Parameters.

[0102] The association of [³H]RTX (60 pM) to VR1 expressed on HEK293cells was rapid: within 10 min the specific binding attainedapproximately 90% of its peak value and it then remained on a plateaubetween 20 min and 60 min of incubation (a single experiment; data notshown). If dissociation was initiated following a 60 min association, itcould be fitted to a 1st order decay curve, yielding a dissociationconstant of 0.12+/−0.02 min-1 (two determinations; data not shown).Based on these preliminary experiments, an incubation period of 60 minwas selected for the equilibrium binding studies.

[0103] [³H]RTX (7-1,000 pM) displayed saturable binding to HEK293/VR1cells (FIG. 1A). The half-maximal binding occurred at 84+/−11 pM(mean+/−S.E.M.; 4 determinations); at the K_(d), non-specific bindingrepresented approximately 20% of the total binding (not shown). Thesaturation binding curve was sigmoidal, indicating positivecooperativity (FIG. 1B). A fit to the allosteric Hill equation yielded acooperativity index of 2.1+/−0.2 (mean+/−S.E.M.; 4 determinations). Thisbinding behavior results in a convex Scatchard plot (FIG. 1C). TheB_(max) value was 250+/−24 fmol/10⁶ cells (mean+/−S.E.M.; 4determinations), corresponding to a receptor density of 1.5×10⁵ bindingsites per cell. CHO/VR1 cells bound RTX with similar affinity (a K_(d)of 103+/−13 pM; mean+/−range; 2 determinations) and cooperativity values(a Hill number of 1.9+/−0.1; mean+/−range; 2 experiments). The maximalreceptor density was, however, approximately two-fold higher than in theHEK293/VR1 cells (470+/−30 fmol/10⁶ cells; mean+/−range; 2determinations) (FIG. 1C). The VR1-transfected cells lines bound RTXwith parameters similar not only to each other but also to rat DRGmembranes expressing native vanilloid receptors (FIG. 2). DRG membranesbound [³H]RTX with a K_(d) of 70+/−10 pM and a B_(max) of 290+/−10fmol/mg protein (mean+/−range; 2 determinations); the cooperativityindex was 1.9+/−2 (mean+/−range; 2 determinations).

[0104] Although the CHO/VR1 cells provided a higher level of specificbinding, HEK293/VR1 cells were chosen for detailed further analysis fora better comparison with the literature (Caterina et al., 1997; Tominagaet al., 1998).

[0105] Vanilloid Agonists and the Antagonist Capsazepine Inhibit [³H]RTXBinding by VR1-Transfected Mammalian Cells and DRG Membranes,Respectively, with Similar Affinities.

[0106] For the pharmacological characterization of the RTX-recognitionsite on rat VR1 expressed in HEK293 cells, four agonists (olvanil,capsaicin, isovelleral, and scutigeral) and an antagonist (capsazepine)were selected (FIG. 3). K_(i) values of the agonists were the following:olvanil, 0.4+/−0.1 μM (n=4); capsaicin, 4.0+/−0.8 μM (n=6); isovelleral,20+/−4 μM (n=3); and scutigeral, 18+/−3 μM (n=3); all values aremean+/−S.E.M. The competitive antagonist capsazepine inhibited [³H]RTXbinding with a K_(i) of 6.2+/−0.7 μM (mean+/−S.E.M.; 5 experiments).These K_(i) values are similar to those determined using rat DRGmembranes: olvanil, 0.3+/−0.1 μM; capsaicin, 2.5+/−1.1 μM; isovelleral,24+/−4 μM; scutigeral, 21+/−3 μM; and capsazepine, 8.6+/−3.5 μM(mean+/−S.E.M.; 3 experiments; Table III) The binding affinities ofolvanil, capsaicin and capsazepine were also determined using CHO/VR1cells: K_(i) values were 0.26+/−0.5 μM, 1.3+/−0.4 μM, and 6.6+/−1.4 μM,respectively (mean+/−range; 2 determinations; TABLE III) TABLE III Ratdorsal root ganglion neurons CHO/VR1 cells Binding ⁴⁵Ca²⁺ - BindingCa²⁺ - affinity uptake affinity mobilization Ligand (nM) (EC₅₀, nM)*(nM) (EC₅₀, nM) Resinifera- 0.07  1 0.13  1.4 toxin capsaicin 2,500 3401,700  38 olvanil 300 170** 260 216 capsazepine*** 8,600 271 5,100 140(1,100) Ruthenium no effect 790 no effect 210 red

[0107] Characterization of rat capsaicin receptor VR1-transfectedmammalian cells in the calcium mobilization assay using variousvanilloid compounds. Capsaicin induced calcium mobilization inHEK293/VR1 cells and CHO/VR1 cells with EC₅₀ values of 82+/−17 nM(mean+/−S.E.M.; n=4) and 38+/−16 nM (mean+/−S.E.M.; n=5), respectively.RTX was more than an order of a magnitude more potent in both celllines; EC₅₀ values were 4.1+/−1.3 nM in HEK293/VR1 cells (mean+/−S.E.M.;n=5), and 1.4+/−0.8 nM in CHO/VR1 cells (mean+/−S.E.M.; n=4). Capsaicinand RTX differed not only in potency in the calcium mobilization assay,but also in the kinetics of the response as shown by FIG. 4. Using 30 nMcapsaicin, a concentration close to the EC₅₀ in CHO/VR1 cells, led torapid calcium mobilization responses: the maximal fluorescence changeoccurred within 30 sec. (30 μM capsaicin) to 50 sec. (3 nM capsaicin).By contrast, RTX-evoked calcium mobilization became detectable onlyafter an initial delay (compare FIG. 4A and FIG. 4B). The calciummobilization response to capsaicin achieved its peak value within 1 min.and then started to decline, suggestive of the development oftachyphylaxis or due to some other aspect of channel gating (FIG. 4A).By contrast, unless high RTX concentrations were used (such as 100 nM, avalue almost 100-fold higher than the EC₅₀), resiniferatoxin applicationresulted in slowly developing but persistent calcium currents. Calciummobilization in response to 1 nM RTX increased steadily over a 3 min.period after challenge, approaching the maximal response evoked by 100nM RTX (FIG. 4B). This difference between the kinetics ofcapsaicin-induced and RTX-induced responses, however, disappeared whenhigh, supramaximal doses were used (30 uM capsaicin or 100 nM RTX;compare FIG. 4A and FIG. 4B). Olvanil evoked the calcium response inCHO/VR1 cell with a potency of 22+/−6 nM (mean+/−S.E.M.; n=7). Thetime-course of the olvanil-induced calcium mobilization response wassimilar to that triggered by capsaicin (not shown). When 25 nM capsaicinwas administered to evoke calcium mobilization, capsazepine inhibitedthis response with an IC₅₀ value of 2.4+/−0.5 uM (mean+/−S.E.M.; n=6).This value was, however, shifted by almost an order of magnitude in thepresence of 1 mg/ml bovine serum albumin to yield an IC₅₀ value of0.33+/−0.03 uM (mean+/−S.E.M.; n=5). For the other vanilloids tested inthis study, the presence or absence of bovine serum albumin had nodetectable influence on the calcium mobilization potency. With an IC₅₀of 210+/−30 NM (mean+/−S.E.M.; n=7), the functional antagonist rutheniumred was similar in potency to capsazepine in ability to prevent calciummobilization by capsaicin.

[0108] A further discussion of these results can be found in Szallasi etal., 1999.

[0109] Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Various modifications of the described modes for carryingout the invention that are obvious to those skilled in the relevant artsare within the scope of the invention. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

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[0111] Bevan, S. and Geppetti, P. (1994) Trends Neurosci. 17: 509-512.

[0112] Caterina, M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A.,Levine, J. D. and Julius, D., (1997) Nature 389: 816-824.

[0113] Caterina, M. J., Rosen, T. A., Tominaga, N., Brake, A. J., andJulius, D. (1999) Nature 398: 436-441.

[0114] Chou, P. Y. and Fasman G. D. (1974) Biochemistry 13: 222-244

[0115] Clapham, D. E. (1996) Neuron 16: 1069-1072.

[0116] Julius, D., Caterina, M., and Brake, A. WIPO-PCT publication No.WO 99/09140.

[0117] Kirshstein, T., Busselberg, D., Treede, R. D. (1997) Neurosci.Lett. 231(1): 33-36.

[0118] Kress, M., Fetzer, S., Reeh, P. W. and Vyklicky (1996) Neurosci.Lett. 211: 5-8.

[0119] Kress, M. and Reeh, P. W. (1996) in Neurobiology of Nociceptors(Cervero, F. and Belmonte, C., eds.), 258-297, Oxford University Press.

[0120] Muench, G., Walker, P., Shine, J. and Herzog, H. (1995) Recept.Channels 3(4): 291-297.

[0121] Schulz, G. E. and Schirmer, R. H. (1990) in Principles of ProteinStructure, Charles Cantor, editor, p. 14-16, Springer-Verlag, NY

[0122] Oh, U., Hwang, S. W. and Kim, D. (1996) J. Neurosci. 16:1659-1667.

[0123] Szallasi, A. and Blumberg, P. M. (1989) Neuroscience 30: 515-520.

[0124] Szallasi, A., Lewin, N. E. and Blumberg, P. M. (1992) J.Pharmacol. Exp. Ter. 262: 883-888.

[0125] Szallasi, Lewin, N. E. and Blumberg, P. M. (1993) J. Pharmacol.Exp. Ther. 266: 678-683.

[0126] Szallasi, A., Blumberg, P. M., Annicelli, L. L., Krause, J., andCortright, D. N. (1999) Mol. Pharmacol. 56(3): 581-7.

[0127] Szolcsanyi, J and Jancso-Gabor, A. (1975) Drug Res. 25:1877-1881.

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[0129] Tominaga, M., Caterain, M. J., Malmber, A. B., Rosen, T. A.,Gilbert, H., Skinner, K., Raumann, B. E., Basbaum, A. I. and Julius, D.(1998) Neuron 21: 531-543.

[0130] Walpole, C. S. J. and Wrigglesworth, R. (1993) in Capsaicin inthe Study of Pain (Wood, J. N., ed.) pp. 63-82, Academic Press, SanDiego.

[0131] Zeilhofer, H. U., Kress, M. and Swandulla, D. (1997) J. Physiol.503: 67-78.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 13 <210> SEQ ID NO 1<211> LENGTH: 4203 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 ccgaagggcg tccacagcga ctcctgctat gcagggcagc tgctgccagggccgggcccg 60 ggaccccacg gaggcgggga gaccactctt ctcccacacg agcccagctctcccttcgag 120 tagcaaccgc cttcaagctc acaagcaccc gtgggcctgg ggtgtgcctgcgtctagctg 180 gttgcacact gggccacaga ggatccagca aggatgaaga aatggagcagcacagacttg 240 ggggcagctg cggacccact ccaaaaggac acctgcccag accccctggatggagaccct 300 aactccaggc cacctccagc caagccccag ctctccacgg ccaagagccgcacccggctc 360 tttgggaagg gtgactcgga ggaggctttc ccggtggatt gccctcacgaggaaggtgag 420 ctggactcct gcccgaccat cacagtcagc cctgttatca ccatccagaggccaggagac 480 ggccccaccg gtgccaggct gctgtcccag gactctgtcg ccgccagcaccgagaagacc 540 ctcaggctct atgatcgcag gagtatcttt gaagccgttg ctcagaataactgccaggat 600 ctggagagcc tgctgctctt cctgcagaag agcaagaagc acctcacagacaacgagttc 660 aaagaccctg agacagggaa gacctgtctg ctgaaagcca tgctcaacctgcacgacgga 720 cagaacacca ccatccccct gctcctggag atcgcgcggc aaacggacagcctgaaggag 780 cttgtcaacg ccagctacac ggacagctac tacaagggcc agacagcactgcacatcgcc 840 atcgagagac gcaacatggc cctggtgacc ctcctggtgg agaacggagcagacgtccag 900 gctgcggccc atggggactt ctttaagaaa accaaagggc ggcctggattctacttcggt 960 gaactgcccc tgtccctggc cgcgtgcacc aaccagctgg gcatcgtgaagttcctgctg 1020 cagaactcct ggcagacggc cgacatcagc gccagggact cggtgggcaacacggtgctg 1080 cacgccctgg tggaggtggc cgacaacacg gccgacaaca cgaagtttgtgacgagcatg 1140 tacaatgaga ttctgatgct gggggccaaa ctgcacccga cgctgaagctggaggagctc 1200 accaacaaga agggaatgac gccgctggct ctggcagctg ggaccgggaagatcggggtc 1260 ttggcctata ttctccagcg ggagatccag gagcccgagt gcaggcacctgtccaggaag 1320 ttcaccgagt gggcctacgg gcccgtgcac tcctcgctgt acgacctgtcctgcatcgac 1380 acctgcgaga agaactcggt gctggaggtg atcgcctaca gcagcagcgagacccctaat 1440 cgccacgaca tgctcttggt ggagccgctg aaccgactcc tgcaggacaagtgggacaga 1500 ttcgtcaagc gcatcttcta cttcaacttc ctggtctact gcctgtacatgatcatcttc 1560 accatggctg cctactacag gcccgtggat ggcttgcctc cctttaagatggaaaaaatt 1620 ggagactatt tccgagttac tggagagatc ctgtctgtgt taggaggagtctacttcttt 1680 ttccgaggga ttcagtattt cctgcagagg cggccgtcga tgaagaccctgtttgtggac 1740 agctacagtg agatgctttt ctttctgcag tcactgttca tgctggccaccgtggtgctg 1800 tacttcagcc acctcaagga gtatgtggct tccatggtat tctccctggccttgggctgg 1860 accaacatgc tctactacac ccgcggtttc cagcagatgg gcatctatgccgtcatgata 1920 gagaagatga tcctgagaga cctgtgccgt ttcatgtttg tctacatcgtcttcttgttc 1980 gggttttcca cagcggtggt gacgctgatt gaagacggga agaatgactccctgccgtct 2040 gagtccacgt cgcacaggtg gcgggggcct gcctgcaggc cccccgatagctcctacaac 2100 agcctgtact ccacctgcct ggagctgttc aagttcacca tcggcatgggcgacctggag 2160 ttcactgaga actatgactt caaggctgtc ttcatcatcc tgctgctggcctatgtaatt 2220 ctcacctaca tcctcctgct caacatgctc atcgccctca tgggtgagactgtcaacaag 2280 atcgcacagg agagcaagaa catctggaag ctgcagagag ccatcaccatcctggacacg 2340 gagaagagct tccttaagtg catgaggaag gccttccgct caggcaagctgctgcaggtg 2400 gggtacacac ctgatggcaa ggacgactac cggtggtgct tcagggtggacgaggtgaac 2460 tggaccacct ggaacaccaa cgtgggcatc atcaacgaag acccgggcaactgtgagggc 2520 gtcaagcgca ccctgagctt ctccctgcgg tcaagcagag tttcaggcagacactggaag 2580 aactttgccc tggtccccct tttaagagag gcaagtgctc gagataggcagtctgctcag 2640 cccgaggaag tttatctgcg acagttttca gggtctctga agccagaggacgctgaggtc 2700 ttcaagagtc ctgccgcttc cggggagaag tgaggacgtc acgcagacagcactgtcaac 2760 actgggcctt aggagacccc gttgccacgg ggggctgctg agggaacaccagtgctctgt 2820 cagcagcctg gcctggtctg tgcctgccca gcatgttccc aaatctgtgctggacaagct 2880 gtgggaagcg ttcttggaag catggggagt gatgtacatc caaccgtcattgtccccaag 2940 tgaatctcct aacagacttt caggttttta ctcactttac taaacagtgtggatggtcag 3000 tctctactgg gacatgttag gcccttgttt tctttgattt tattcttttttttgagacag 3060 aatttcactc ttctcgccca ggctggaatg cagtggcaca attttggctccctgcaacct 3120 ccgcctcctg gattccagca attctcctgc ctcggcttcc caagtagctgggattacagg 3180 cacgtgccac catgtctggc taattttttg tattttttta atagatatggggtttcgcca 3240 tgttggccag gctggtctcg aactcctgac ctcaggtgat ccgcccacctcggcctccca 3300 aagtgctggg attacaggtg tgagcctcca cacctggctg ttttctttgattttattctt 3360 tttttttttt ttctgtgaga cagagtttca ctcttgttgc ccaggctggagtgcagtggt 3420 gtgatcttgg ctcactgcaa cctctgcctc ccgggttcaa gcgattcttctgcttcagtc 3480 tcccaagtag cttggattac aggtgagcac taccacgccc ggctaatttttgtattttta 3540 atagagacgg ggtttcacca tgttggccag gctggtctcg aactcttgacctcaggtgat 3600 ctgcccgcct tggcctccca aagtgctggg attacaggtg tgagccgctgcgctcggcct 3660 tctttgattt tatattatta ggagcaaaag taaatgaagc ccaggaaaacacctttggga 3720 acaaactctt cctttgatgg aaaatgcaga ggcccttcct ctctgtgccgtgcttgctcc 3780 tcttacctgc ccgggtggtt tgggggtgtt ggtgtttcct ccctggagaagatgggggag 3840 gctgtcccac tcccagctct ggcagaatca agctgttgca gcagtgccttcttcatcctt 3900 ccttacgatc aatcacagtc tccagaagat cagctcaatt gctgtgcaggttaaaactac 3960 agaaccacat cccaaaggta cctggtaaga atgtttgaaa gatcttccatttctaggaac 4020 cccagtcctg cttctccgca atggcacatg cttccactcc atccatactggcatcctcaa 4080 ataaacagat atgtatwcat ataaaaaaaa aaaaaaaaaa aaaaaaaaactcgagagtac 4140 ttctagagcg gccgcgggcc catcgatttt ccacccgggt ggggtaccaggtaaggtgcc 4200 aac 4203 <210> SEQ ID NO 2 <211> LENGTH: 4182 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 tgctagtgcagggcagctgc tgccagggcc gggcccggga ccccacggag gcggggagac 60 cactcttctcccacacgagc ccagctctcc cttcgagtag caaccgcctt caagctcaca 120 agcacccgtgggcctggggt gtgcctgcgt ctagctggtt gcacactggg ccacagagga 180 tccagcaaggatgaagaaat ggagcagcac agacttgggg gcagctgcgg acccactcca 240 aaaggacacctgcccagacc ccctggatgg agaccctaac tccaggccac ctccagccaa 300 gccccagctctccacggcca agagccgcac ccggctcttt gggaagggtg actcggagga 360 ggctttcccggtggattgcc ctcacgagga aggtgagctg gactcctgcc cgaccatcac 420 agtcagccctgttatcacca tccagaggcc aggagacggc cccaccggtg ccaggctgct 480 gtcccaggactctgtcgccg ccagcaccga gaagaccctc aggctctatg atcgcaggag 540 tatctttgaagccgttgctc agaataactg ccaggatctg gagagcctgc tgctcttcct 600 gcagaagagcaagaagcacc tcacagacaa cgagttcaaa gaccctgaga cagggaagac 660 ctgtctgctgaaagccatgc tcaacctgca cgacggacag aacaccacca tccccctgct 720 cctggagatcgcgcggcaaa cggacagcct gaaggagctt gtcaacgcca gctacacgga 780 cagctactacaagggccaga cagcactgca catcgccatc gagagacgca acatggccct 840 ggtgaccctcctggtggaga acggagcaga cgtccaggct gcggcccatg gggacttctt 900 taagaaaaccaaagggcggc ctggattcta cttcggtgaa ctgcccctgt ccctggccgc 960 gtgcaccaaccagctgggca tcgtgaagtt cctgctgcag aactcctggc agacggccga 1020 catcagcgccagggactcgg tgggcaacac ggtgctgcac gccctggtgg aggtggccga 1080 caacacggccgacaacacga agtttgtgac gagcatgtac aatgagattc tgatgctggg 1140 ggccaaactgcacccgacgc tgaagctgga ggagctcacc aacaagaagg gaatgacgcc 1200 gctggctctggcagctggga ccgggaagat cggggtcttg gcctatattc tccagcggga 1260 gatccaggagcccgagtgca ggcacctgtc caggaagttc accgagtggg cctacgggcc 1320 cgtgcactcctcgctgtacg acctgtcctg catcgacacc tgcgagaaga actcggtgct 1380 ggaggtgatcgcctacagca gcagcgagac ccctaatcgc cacgacatgc tcttggtgga 1440 gccgctgaaccgactcctgc aggacaagtg ggacagattc gtcaagcgca tcttctactt 1500 caacttcctggtctactgcc tgtacatgat catcttcacc atggctgcct actacaggcc 1560 cgtggatggcttgcctccct ttaagatgga aaaaattgga gactatttcc gagttactgg 1620 agagatcctgtctgtgttag gaggagtcta cttctttttc cgagggattc agtatttcct 1680 gcagaggcggccgtcgatga agaccctgtt tgtggacagc tacagtgaga tgcttttctt 1740 tctgcagtcactgttcatgc tggccaccgt ggtgctgtac ttcagccacc tcaaggagta 1800 tgtggcttccatggtattct ccctggcctt gggctggacc aacatgctct actacacccg 1860 cggtttccagcagatgggca tctatgccgt catgatagag aagatgatcc tgagagacct 1920 gtgccgtttcatgtttgtct acatcgtctt cttgttcggg ttttccacag cggtggtgac 1980 gctgattgaagacgggaaga atgactccct gccgtctgag tccacgtcgc acaggtggcg 2040 ggggcctgcctgcaggcccc ccgatagctc ctacaacagc ctgtactcca cctgcctgga 2100 gctgttcaagttcaccatcg gcatgggcga cctggagttc actgagaact atgacttcaa 2160 ggctgtcttcatcatcctgc tgctggccta tgtaattctc acctacatcc tcctgctcaa 2220 catgctcatcgccctcatgg gtgagactgt caacaagatc gcacaggaga gcaagaacat 2280 ctggaagctgcagagagcca tcaccatcct ggacacggag aagagcttcc ttaagtgcat 2340 gaggaaggccttccgctcag gcaagctgct gcaggtgggg tacacacctg atggcaagga 2400 cgactaccggtggtgcttca gggtggacga ggtgaactgg accacctgga acaccaacgt 2460 gggcatcatcaacgaagacc cgggcaactg tgagggcgtc aagcgcaccc tgagcttctc 2520 cctgcggtcaagcagagttt caggcagaca ctggaagaac tttgccctgg tccccctttt 2580 aagagaggcaagtgctcgag ataggcagtc tgctcagccc gaggaagttt atctgcgaca 2640 gttttcagggtctctgaagc cagaggacgc tgaggtcttc aagagtcctg ccgcttccgg 2700 ggagaagtgaggacgtcacg cagacagcac tgtcaacact gggccttagg agaccccgtt 2760 gccacggggggctgctgagg gaacaccagt gctctgtcag cagcctggcc tggtctgtgc 2820 ctgcccagcatgttcccaaa tctgtgctgg acaagctgtg ggaagcgttc ttggaagcat 2880 ggggagtgatgtacatccaa ccgtcactgt ccccaagtga atctcctaac agactttcag 2940 gtttttactcactttactaa acagtgtgga tggtcagtct ctactgggac atgttaggcc 3000 cttgttttctttgattttat tctttttttt gagacagaat ttcactcttc tcgcccaggc 3060 tggaatgcagtggcacaatt ttggctccct gcaacctccg cctcctggat tccagcaatt 3120 ctcctgcctcggcttcccaa gtagctggga ttacaggcac gtgccaccat gtctggctaa 3180 ttttttgtatttttttaata gatatggggt ttcgccatgt tggccaggct ggtctcgaac 3240 tcctgacctcaggtgatccg cccacctcgg cctcccaaag tgctgggatt acaggtgtga 3300 gcctccacacctggctgttt tctttgattt tattcttttt ttttttttct gtgagacaga 3360 gtttcactcttgttgcccag gctggagtgc agtggtgtga tcttggctca ctgcaacctc 3420 tgcctcccgggttcaagcga ttcttctgct tcagtctccc aagtagcttg gattacaggt 3480 gagcactaccacgcccggct aatttttgta tttttaatag agacggggtt tcaccatgtt 3540 ggccaggctggtctcgaact cttgacctcg ggtgatctgc ccgccttggc ctcccaaagt 3600 gctgggattacaggtgtgag ccgctgcgct cggccttctt tgattttata ttattaggag 3660 caaaagtaaatgaagcccag gaaaacacct ttgggaacaa actcttcctt tgatggaaaa 3720 tgcagaggcccttcctctct gtgccgtgct tgctcctctt acctgcccgg gtggtttggg 3780 ggtgttggtgtttcctccct ggagaagatg ggggaggctg tcccactccc agctctggca 3840 gaatcaagctgttgcagcag tgccttcttc atccttcctt acgatcaatc acagtctcca 3900 gaagatcagctcaattgctg tgcaggttaa aactacagaa ccacatccca aaggtacctg 3960 gtaagaatgtttgaaagatc ttccatttct aggaacccca gtcctgcttc tccgcaatgg 4020 cacatgcttccactccatcc atactggcat cctcaaataa acagatatgt atacatataa 4080 aaaaaaaaaaaaaaaaaaaa aaaaactcga gagtacttct agagcggccg cgggcccatc 4140 gattttccacccgggtgggg taccaggtaa gtgtacccaa tc 4182 <210> SEQ ID NO 3 <211> LENGTH:4171 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3gcagagtgtg cagtatagat tcagcgtttg tcgactgact gaatgatagc acaatccagg 60ctgcttcctg ttggctgggt ttggttggac tgggacccgt cagaggaaaa ggcaacgccg 120ctgacaaaga acattgccga aaggttcatg ggaggctccg gctaacaggt tgcacactgg 180gccacagagg atccagcaag gatgaagaaa tggagcagca cagacttggg ggcagctgcg 240gacccactcc aaaaggacac ctgcccagac cccctggatg gagaccctaa ctccaggcca 300cctccagcca agccccagct ctccacggcc aagagccgca cccggctctt tgggaagggt 360gactcggagg aggctttccc ggtggattgc cctcacgagg aaggtgagct ggactcctgc 420ccgaccatca cagtcagccc tgttatcacc atccagaggc caggagacgg ctccaccggt 480gccaggctgc tgtcccagga ctctgtcgcc gccagcaccg agaagaccct caggctctat 540gatcgcagga gtatctttga agccgttgct cagaataact gccaggatct ggagagcctg 600ctgctcttcc tgcagaagag caagaagcac ctcacagaca acgagttcaa agaccctgag 660acagggaaga cctgtctgct gaaagccatg ctcaacctgc atgacggaca gaacaccacc 720atccccctgc tcctggagat cgcgcggcaa acggacagcc tgaaggagct tgtcaacgcc 780agctacacgg acagctacta caagggccag acagcactgc acatcgccat cgagagacgc 840aacatggccc tggtgaccct cctggtggag aacggagcag acgtccaggc tgcggcccat 900ggggacttct ttaagaaaac caaagggcgg cctggattct acttcggtga actgcccctg 960tccctggccg cgtgcaccaa ccagctgggc atcgtgaagt tcctgctgca gaactcctgg 1020cagacggccg acatcagcgc cagggactcg gtgggcaaca cggtgctgca cgccctggtg 1080gaggtggccg acaacacggc cgacaacacg aagtttgtga cgagcatgta caatgagatt 1140ctgatcctgg gggccaaact gcacccgacg ctgaagctgg aggagctcac caacaagaag 1200ggaatgacgc cgctggctct ggcagctggg accgggaaga tcggggtctt ggcctatatt 1260ctccagcggg agatccagga gcccgagtgc aggcacctgt ccaggaagtt caccgagtgg 1320gcctacgggc ccgtgcactc ctcgctgtac gacctgtcct gcatcgacac ctgcgagaag 1380aactcggtgc tggaggtgat cgcctacagc agcagcgaga cccctaatcg ccacgacatg 1440ctcttggtgg agccgctgaa ccgactcctg caggacaagt gggacagatt cgtcaagcgc 1500atcttctact tcaacttcct ggtctactgc ctgtacatga tcatcttcac catggctgcc 1560tactacaggc ccgtggatgg cttgcctccc tttaagatgg aaaaaattgg agactatttc 1620cgagttactg gagagatcct gtctgtgtta ggaggagtct acttcttttt ccgagggatt 1680cagtatttcc tgcaggcggc cgtcgatgaa gaccctgttt gtggacagct acagtgagat 1740gcttttcttt ctgcagtcac tgttcatgct ggccaccgtg gtgctgtact tcagccacct 1800caaggagtat gtggcttcca tggtattctc cctggccttg ggctggacca acatgctcta 1860ctacacccgc ggtttccagc agatgggcat ctatgccgtc atgatagaga agatgatcct 1920gagagacctg tgccgtttca tgtttgtcta catcgtcttc ttgttcgggt tttccacagc 1980ggtggtgacg ctgattgaag acgggaagaa tgactccctg ccgtctgagt ccacgtcgca 2040caggtggcgg gggcctgcct gcaggccccc cgatagctcc tacaacagcc tgtactccac 2100ctgcctggag ctgttcaagt tcaccatcgg catgggcgac ctggagttca ctgagaacta 2160tgacttcaag gctgtcttca tcatcctgct gctggcctat gtaattctca cctacatcct 2220cctgctcaac atgctcatcg ccctcatggg tgagactgtc aacaagatcg cacaggagag 2280caagaacatc tggaagctgc agagagccat caccatcctg gacacggaga agagcttcct 2340taagtgcatg aggaaggcct tccgctcagg caagctgctg caggtggggt acacacctga 2400tggcaaggac gactaccggt ggtgcttcag ggtggacgag gtgaactgga ccacctggaa 2460caccaacgtg ggcatcatca acgaagaccc gggcaactgt gagggcgtca agcgcaccct 2520gagcttctcc ctgcggtcaa gcagagtttc aggcagacac tggaagaact ttgccctggt 2580ccccctttta agagaggcaa gtgctcgaga taggcagtct gctcagcccg aggaagttta 2640tctgcgacag ttttcagggt ctctgaagcc agaggacgct gaggtcttca agagtcctgc 2700cgcttccggg gagaagtgag gacgtcacgc agacagcact gtcaacactg ggccttagga 2760gaccccgttg ccacgggggg ctgctgaggg aacaccagtg ctctgtcagc agcctggcct 2820ggtctgtgcc tgcccagcat gttcccaaat ctgtgctgga caagctgtgg gaagcgttct 2880tggaagcatg gggagtgatg tacatccaac cgtcactgtc cccaagtgaa tctcctaaca 2940gactttcagg tttttactca ctttactaaa cagtttggat ggtcagtctc tactgggaca 3000tgttaggccc ttgttttctt tgattttatt cttttttttg agacagaatt tcactcttct 3060cacccaggct ggaatgcagt ggcacaattt tggctccctg caacctccgc ctcctggatt 3120ccagcaattc tcctgcctcg gcttcccaag tagctgggat tacaggcacg tgccaccatg 3180tctggctaat tttttgtatt tttttaatag atatggggtt tcgccatgtt ggccaggctg 3240gtctcgaact cctgacctca ggtgatccgc ccacctcggc ctcccaaagt gctgggatta 3300caggtgtgag cctccacacc tggctgtttt ctttgatttt attctttttt tttttttctg 3360tgagacagag tttcactctt gttgcccagg ctggagtgca gtggtgtgat cttggctcac 3420tgcaacctct gcctcccggg ttcaagcgat tcttctgctt cagtctccca agtagcttgg 3480attacaggtg agcactacca cgcccggcta atttttgtat ttttaataga gacggggttt 3540caccatgttg gccaggctgg tctcgaactc ttgacctcag gtgatctgcc cgccttggcc 3600tcccaaagtg ctgggattac aggtgtgagc tgctgcgctc ggccttcttt gattttatat 3660tattaggagc aaaagtaaat gaagcccagg aaaacacctt tgggaacaaa ctcttccttt 3720gatggaaaat gcagaggccc ttcctctctg tgccgtgctt gctcctctta cctgcccggg 3780tggtttgggg gtgttggtgt ttcctccctg gagaagatgg gggaggctgt cccactccca 3840gctctggcag aatcaagctg ttgcagcagt gccttcttca tccttcctta cgatcaatca 3900cagtctccag aagatcagct caattgctgt gcaggttaaa actacagaac cacatcccaa 3960aggtacctgg taagaatgtt tgaaagatct tccatttcta ggaaccccag tcctgcttct 4020ccgcaatggc acatgcttcc actccatcca tactggcatc ctcaaataaa cagatatgta 4080tacataaaaa aaaaaaaaaa aaactcgaga gtacttctag agcggccgcg ggcccatcga 4140ttttccaccc gggtggggta ccaggtaagt g 4171 <210> SEQ ID NO 4 <211> LENGTH:839 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: TRANSMEM <222> LOCATION: (434)..(455) <223> OTHER INFORMATION:TM1 <220> FEATURE: <221> NAME/KEY: TRANSMEM <222> LOCATION: (480)..(495)<223> OTHER INFORMATION: TM2 <220> FEATURE: <221> NAME/KEY: TRANSMEM<222> LOCATION: (510)..(530) <223> OTHER INFORMATION: TM3 <220> FEATURE:<221> NAME/KEY: TRANSMEM <222> LOCATION: (543)..(569) <223> OTHERINFORMATION: TM4 <220> FEATURE: <221> NAME/KEY: TRANSMEM <222> LOCATION:(577)..(596) <223> OTHER INFORMATION: TM5 <220> FEATURE: <221> NAME/KEY:TRANSMEM <222> LOCATION: (656)..(684) <223> OTHER INFORMATION: TM6 <400>SEQUENCE: 4 Met Lys Lys Trp Ser Ser Thr Asp Leu Gly Ala Ala Ala Asp ProLeu 1 5 10 15 Gln Lys Asp Thr Cys Pro Asp Pro Leu Asp Gly Asp Pro AsnSer Arg 20 25 30 Pro Pro Pro Ala Lys Pro Gln Leu Ser Thr Ala Lys Ser ArgThr Arg 35 40 45 Leu Phe Gly Lys Gly Asp Ser Glu Glu Ala Phe Pro Val AspCys Pro 50 55 60 His Glu Glu Gly Glu Leu Asp Ser Cys Pro Thr Ile Thr ValSer Pro 65 70 75 80 Val Ile Thr Ile Gln Arg Pro Gly Asp Gly Pro Thr GlyAla Arg Leu 85 90 95 Leu Ser Gln Asp Ser Val Ala Ala Ser Thr Glu Lys ThrLeu Arg Leu 100 105 110 Tyr Asp Arg Arg Ser Ile Phe Glu Ala Val Ala GlnAsn Asn Cys Gln 115 120 125 Asp Leu Glu Ser Leu Leu Leu Phe Leu Gln LysSer Lys Lys His Leu 130 135 140 Thr Asp Asn Glu Phe Lys Asp Pro Glu ThrGly Lys Thr Cys Leu Leu 145 150 155 160 Lys Ala Met Leu Asn Leu His AspGly Gln Asn Thr Thr Ile Pro Leu 165 170 175 Leu Leu Glu Ile Ala Arg GlnThr Asp Ser Leu Lys Glu Leu Val Asn 180 185 190 Ala Ser Tyr Thr Asp SerTyr Tyr Lys Gly Gln Thr Ala Leu His Ile 195 200 205 Ala Ile Glu Arg ArgAsn Met Ala Leu Val Thr Leu Leu Val Glu Asn 210 215 220 Gly Ala Asp ValGln Ala Ala Ala His Gly Asp Phe Phe Lys Lys Thr 225 230 235 240 Lys GlyArg Pro Gly Phe Tyr Phe Gly Glu Leu Pro Leu Ser Leu Ala 245 250 255 AlaCys Thr Asn Gln Leu Gly Ile Val Lys Phe Leu Leu Gln Asn Ser 260 265 270Trp Gln Thr Ala Asp Ile Ser Ala Arg Asp Ser Val Gly Asn Thr Val 275 280285 Leu His Ala Leu Val Glu Val Ala Asp Asn Thr Ala Asp Asn Thr Lys 290295 300 Phe Val Thr Ser Met Tyr Asn Glu Ile Leu Met Leu Gly Ala Lys Leu305 310 315 320 His Pro Thr Leu Lys Leu Glu Glu Leu Thr Asn Lys Lys GlyMet Thr 325 330 335 Pro Leu Ala Leu Ala Ala Gly Thr Gly Lys Ile Gly ValLeu Ala Tyr 340 345 350 Ile Leu Gln Arg Glu Ile Gln Glu Pro Glu Cys ArgHis Leu Ser Arg 355 360 365 Lys Phe Thr Glu Trp Ala Tyr Gly Pro Val HisSer Ser Leu Tyr Asp 370 375 380 Leu Ser Cys Ile Asp Thr Cys Glu Lys AsnSer Val Leu Glu Val Ile 385 390 395 400 Ala Tyr Ser Ser Ser Glu Thr ProAsn Arg His Asp Met Leu Leu Val 405 410 415 Glu Pro Leu Asn Arg Leu LeuGln Asp Lys Trp Asp Arg Phe Val Lys 420 425 430 Arg Ile Phe Tyr Phe AsnPhe Leu Val Tyr Cys Leu Tyr Met Ile Ile 435 440 445 Phe Thr Met Ala AlaTyr Tyr Arg Pro Val Asp Gly Leu Pro Pro Phe 450 455 460 Lys Met Glu LysIle Gly Asp Tyr Phe Arg Val Thr Gly Glu Ile Leu 465 470 475 480 Ser ValLeu Gly Gly Val Tyr Phe Phe Phe Arg Gly Ile Gln Tyr Phe 485 490 495 LeuGln Arg Arg Pro Ser Met Lys Thr Leu Phe Val Asp Ser Tyr Ser 500 505 510Glu Met Leu Phe Phe Leu Gln Ser Leu Phe Met Leu Ala Thr Val Val 515 520525 Leu Tyr Phe Ser His Leu Lys Glu Tyr Val Ala Ser Met Val Phe Ser 530535 540 Leu Ala Leu Gly Trp Thr Asn Met Leu Tyr Tyr Thr Arg Gly Phe Gln545 550 555 560 Gln Met Gly Ile Tyr Ala Val Met Ile Glu Lys Met Ile LeuArg Asp 565 570 575 Leu Cys Arg Phe Met Phe Val Tyr Ile Val Phe Leu PheGly Phe Ser 580 585 590 Thr Ala Val Val Thr Leu Ile Glu Asp Gly Lys AsnAsp Ser Leu Pro 595 600 605 Ser Glu Ser Thr Ser His Arg Trp Arg Gly ProAla Cys Arg Pro Pro 610 615 620 Asp Ser Ser Tyr Asn Ser Leu Tyr Ser ThrCys Leu Glu Leu Phe Lys 625 630 635 640 Phe Thr Ile Gly Met Gly Asp LeuGlu Phe Thr Glu Asn Tyr Asp Phe 645 650 655 Lys Ala Val Phe Ile Ile LeuLeu Leu Ala Tyr Val Ile Leu Thr Tyr 660 665 670 Ile Leu Leu Leu Asn MetLeu Ile Ala Leu Met Gly Glu Thr Val Asn 675 680 685 Lys Ile Ala Gln GluSer Lys Asn Ile Trp Lys Leu Gln Arg Ala Ile 690 695 700 Thr Ile Leu AspThr Glu Lys Ser Phe Leu Lys Cys Met Arg Lys Ala 705 710 715 720 Phe ArgSer Gly Lys Leu Leu Gln Val Gly Tyr Thr Pro Asp Gly Lys 725 730 735 AspAsp Tyr Arg Trp Cys Phe Arg Val Asp Glu Val Asn Trp Thr Thr 740 745 750Trp Asn Thr Asn Val Gly Ile Ile Asn Glu Asp Pro Gly Asn Cys Glu 755 760765 Gly Val Lys Arg Thr Leu Ser Phe Ser Leu Arg Ser Ser Arg Val Ser 770775 780 Gly Arg His Trp Lys Asn Phe Ala Leu Val Pro Leu Leu Arg Glu Ala785 790 795 800 Ser Ala Arg Asp Arg Gln Ser Ala Gln Pro Glu Glu Val TyrLeu Arg 805 810 815 Gln Phe Ser Gly Ser Leu Lys Pro Glu Asp Ala Glu ValPhe Lys Ser 820 825 830 Pro Ala Ala Ser Gly Glu Lys 835 <210> SEQ ID NO5 <211> LENGTH: 511 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: TRANSMEM <222> LOCATION: (434)..(455) <223>OTHER INFORMATION: TM1 <220> FEATURE: <221> NAME/KEY: TRANSMEM <222>LOCATION: (480)..(495) <223> OTHER INFORMATION: TM2 <400> SEQUENCE: 5Met Lys Lys Trp Ser Ser Thr Asp Leu Gly Ala Ala Ala Asp Pro Leu 1 5 1015 Gln Lys Asp Thr Cys Pro Asp Pro Leu Asp Gly Asp Pro Asn Ser Arg 20 2530 Pro Pro Pro Ala Lys Pro Gln Leu Ser Thr Ala Lys Ser Arg Thr Arg 35 4045 Leu Phe Gly Lys Gly Asp Ser Glu Glu Ala Phe Pro Val Asp Cys Pro 50 5560 His Glu Glu Gly Glu Leu Asp Ser Cys Pro Thr Ile Thr Val Ser Pro 65 7075 80 Val Ile Thr Ile Gln Arg Pro Gly Asp Gly Ser Thr Gly Ala Arg Leu 8590 95 Leu Ser Gln Asp Ser Val Ala Ala Ser Thr Glu Lys Thr Leu Arg Leu100 105 110 Tyr Asp Arg Arg Ser Ile Phe Glu Ala Val Ala Gln Asn Asn CysGln 115 120 125 Asp Leu Glu Ser Leu Leu Leu Phe Leu Gln Lys Ser Lys LysHis Leu 130 135 140 Thr Asp Asn Glu Phe Lys Asp Pro Glu Thr Gly Lys ThrCys Leu Leu 145 150 155 160 Lys Ala Met Leu Asn Leu His Asp Gly Gln AsnThr Thr Ile Pro Leu 165 170 175 Leu Leu Glu Ile Ala Arg Gln Thr Asp SerLeu Lys Glu Leu Val Asn 180 185 190 Ala Ser Tyr Thr Asp Ser Tyr Tyr LysGly Gln Thr Ala Leu His Ile 195 200 205 Ala Ile Glu Arg Arg Asn Met AlaLeu Val Thr Leu Leu Val Glu Asn 210 215 220 Gly Ala Asp Val Gln Ala AlaAla His Gly Asp Phe Phe Lys Lys Thr 225 230 235 240 Lys Gly Arg Pro GlyPhe Tyr Phe Gly Glu Leu Pro Leu Ser Leu Ala 245 250 255 Ala Cys Thr AsnGln Leu Gly Ile Val Lys Phe Leu Leu Gln Asn Ser 260 265 270 Trp Gln ThrAla Asp Ile Ser Ala Arg Asp Ser Val Gly Asn Thr Val 275 280 285 Leu HisAla Leu Val Glu Val Ala Asp Asn Thr Ala Asp Asn Thr Lys 290 295 300 PheVal Thr Ser Met Tyr Asn Glu Ile Leu Ile Leu Gly Ala Lys Leu 305 310 315320 His Pro Thr Leu Lys Leu Glu Glu Leu Thr Asn Lys Lys Gly Met Thr 325330 335 Pro Leu Ala Leu Ala Ala Gly Thr Gly Lys Ile Gly Val Leu Ala Tyr340 345 350 Ile Leu Gln Arg Glu Ile Gln Glu Pro Glu Cys Arg His Leu SerArg 355 360 365 Lys Phe Thr Glu Trp Ala Tyr Gly Pro Val His Ser Ser LeuTyr Asp 370 375 380 Leu Ser Cys Ile Asp Thr Cys Glu Lys Asn Ser Val LeuGlu Val Ile 385 390 395 400 Ala Tyr Ser Ser Ser Glu Thr Pro Asn Arg HisAsp Met Leu Leu Val 405 410 415 Glu Pro Leu Asn Arg Leu Leu Gln Asp LysTrp Asp Arg Phe Val Lys 420 425 430 Arg Ile Phe Tyr Phe Asn Phe Leu ValTyr Cys Leu Tyr Met Ile Ile 435 440 445 Phe Thr Met Ala Ala Tyr Tyr ArgPro Val Asp Gly Leu Pro Pro Phe 450 455 460 Lys Met Glu Lys Ile Gly AspTyr Phe Arg Val Thr Gly Glu Ile Leu 465 470 475 480 Ser Val Leu Gly GlyVal Tyr Phe Phe Phe Arg Gly Ile Gln Tyr Phe 485 490 495 Leu Gln Ala AlaVal Asp Glu Asp Pro Val Cys Gly Gln Leu Gln 500 505 510 <210> SEQ ID NO6 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:H-FLAG epitope <400> SEQUENCE: 6 Ala Ser Pro Thr Tyr Arg LeuTyr Ser Ala Ser Pro Ala Ser Pro Ala 1 5 10 15 Ser Pro Ala Ser Pro LeuTyr Ser 20 <210> SEQ ID NO 7 <211> LENGTH: 6 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:His6x epitope <400> SEQUENCE: 7 HisHis His His His His 1 5 <210> SEQ ID NO 8 <211> LENGTH: 2633 <212> TYPE:DNA <213> ORGANISM: Rattus sp. <300> PUBLICATION INFORMATION: <301>AUTHORS: Caterina, Michael J. Schumacher, Mark A. Tominaga, MakotoRosen, Tobias A. Levine, Jon D. Julius, David <302> TITLE: The capsaicinreceptor: a heat-activated ion channel in the pain pathway <303>JOURNAL: Nature <304> VOLUME: 389 <306> PAGES: 816-824 <307> DATE: 1997<400> SEQUENCE: 8 ctggaaagga tggaacaacg ggctagctta gactcagagg agtctgagtccccaccccaa 60 gagaactcct gcctggaccc tccagacaga gaccctaact gcaagccacctccagtcaag 120 ccccacatct tcactaccag gagtcgtacc cggctttttg ggaagggtgactcggaggag 180 gcctctcccc tggactgccc ttatgaggaa ggcgggctgg cttcctgccctatcatcact 240 gtcagctctg ttctaactat ccagaggcct ggggatggac ctgccagtgtcaggccgtca 300 tcccaggact ccgtctccgc tggtgagaag cccccgaggc tctatgatcgcaggagcatc 360 ttcgatgctg tggctcagag taactgccag gagctggaga gcctgctgcccttcctgcag 420 aggagcaaga agcgcctgac tgacagcgag ttcaaagacc cagagacaggaaagacctgt 480 ctgctaaaag ccatgctcaa tctgcacaat gggcagaatg acaccatcgctctgctcctg 540 gacgttgccc ggaagacaga cagcctgaag cagtttgtca atgccagctacacagacagc 600 tactacaagg gccagacagc actgcacatt gccattgaac ggcggaacatgacgctggtg 660 accctcttgg tggagaatgg agcagatgtc caggctgcgg ctaacggggacttcttcaag 720 aaaaccaaag ggaggcctgg cttctacttt ggtgagctgc ccctgtccctggctgcgtgc 780 accaaccagc tggccattgt gaagttcctg ctgcagaact cctggcagcctgcagacatc 840 agcgcccggg actcagtggg caacacggtg cttcatgccc tggtggaggtggcagataac 900 acagttgaca acaccaagtt cgtgacaagc atgtacaacg agatcttgatcctgggggcc 960 aaactccacc ccacgctgaa gctggaagag atcaccaaca ggaaggggctcacgccactg 1020 gctctggctg ctagcagtgg gaagatcggg gtcttggcct acattctccagagggagatc 1080 catgaacccg agtgccgaca cctatccagg aagttcaccg aatgggcctatgggccagtg 1140 cactcctccc tttatgacct gtcctgcatt gacacctgtg aaaagaactcggttctggag 1200 gtgatcgctt acagcagcag tgagacccct aaccgtcatg acatgcttctcgtggaaccc 1260 ttgaaccgac tcctacagga caagtgggac agatttgtca agcgcatcttctacttcaac 1320 ttcttcgtct actgcttgta tatgatcatc ttcaccgcgg ctgcctactatcggcctgtg 1380 gaaggcttgc ccccctataa gctgaaaaac accgttgggg actatttccgagtcaccgga 1440 gagatcttgt ctgtgtcagg aggagtctac ttcttcttcc gagggattcaatatttcctg 1500 cagaggcgac catccctcaa gagtttgttt gtggacagct acagtgagatacttttcttt 1560 gtacagtcgc tgttcatgct ggtgtctgtg gtactgtact tcagccaacgcaaggagtat 1620 gtggcttcca tggtgttctc cctggccatg ggctggacca acatgctctactatacccga 1680 ggattccagc agatgggcat ctatgctgtc atgattgaga agatgatcctcagagacctg 1740 tgccggttta tgttcgtcta cctcgtgttc ttgtttggat tttccacagctgtggtgaca 1800 ctgattgagg atgggaagaa taactctctg cctatggagt ccacaccacacaagtgccgg 1860 gggtctgcct gcaagccagg taactcttac aacagcctgt attccacatgtctggagctg 1920 ttcaagttca ccatcggcat gggcgacctg gagttcactg agaactacgacttcaaggct 1980 gtcttcatca tcctgttact ggcctatgtg attctcacct acatccttctgctcaacatg 2040 ctcattgctc tcatgggtga gaccgtcaac aagattgcac aagagagcaagaacatctgg 2100 aagctgcaga gagccatcac catcctggat acagagaaga gcttcctgaagtgcatgagg 2160 aaggccttcc gctctggcaa gctgctgcag gtggggttca ctcctgacggcaaggatgac 2220 taccggtggt gtttcagggt ggacgaggta aactggacta cctggaacaccaatgtgggt 2280 atcatcaacg aggacccagg caactgtgag ggcgtcaagc gcaccctgagcttctccctg 2340 aggtcaggcc gagtttcagg gagaaactgg aagaactttg ccctggttccccttctgagg 2400 gatgcaagca ctcgagatag acatgccacc cagcaggaag aagttcaactgaagcattat 2460 acgggatccc ttaagccaga ggatgctgag gttttcaagg attccatggtcccaggggag 2520 aaataatgga cactatgcag ggatcaatgc ggggtctttg ggtggtctgcttagggaacc 2580 agcagggttg acgttatctg ggtccactct gtgcctgcct aggcacattccta 2633 <210> SEQ ID NO 9 <211> LENGTH: 838 <212> TYPE: PRT <213>ORGANISM: Rattus sp. <300> PUBLICATION INFORMATION: <301> AUTHORS:Caterina, Michael J. Schumacher, Mark A. Tominaga, Makoto Rosen, TobiasA. <302> TITLE: The capsaicin receptor: a heat-activated ion channel inthe pain pathway <303> JOURNAL: Nature <304> VOLUME: 389 <306> PAGES:816-824 <307> DATE: 1997 <400> SEQUENCE: 9 Met Glu Gln Arg Ala Ser LeuAsp Ser Glu Glu Ser Glu Ser Pro Pro 1 5 10 15 Gln Glu Asn Ser Cys LeuAsp Pro Pro Asp Arg Asp Pro Asn Cys Lys 20 25 30 Pro Pro Pro Val Lys ProHis Ile Phe Thr Thr Arg Ser Arg Thr Arg 35 40 45 Leu Phe Gly Lys Gly AspSer Glu Glu Ala Ser Pro Leu Asp Cys Pro 50 55 60 Tyr Glu Glu Gly Gly LeuAla Ser Cys Pro Ile Ile Thr Val Ser Ser 65 70 75 80 Val Leu Thr Ile GlnArg Pro Gly Asp Gly Pro Ala Ser Val Arg Pro 85 90 95 Ser Ser Gln Asp SerVal Ser Ala Gly Glu Lys Pro Pro Arg Leu Tyr 100 105 110 Asp Arg Arg SerIle Phe Asp Ala Val Ala Gln Ser Asn Cys Gln Glu 115 120 125 Leu Glu SerLeu Leu Pro Phe Leu Gln Arg Ser Lys Lys Arg Leu Thr 130 135 140 Asp SerGlu Phe Lys Asp Pro Glu Thr Gly Lys Thr Cys Leu Leu Lys 145 150 155 160Ala Met Leu Asn Leu His Asn Gly Gln Asn Asp Thr Ile Ala Leu Leu 165 170175 Leu Asp Val Ala Arg Lys Thr Asp Ser Leu Lys Gln Phe Val Asn Ala 180185 190 Ser Tyr Thr Asp Ser Tyr Tyr Lys Gly Gln Thr Ala Leu His Ile Ala195 200 205 Ile Glu Arg Arg Asn Met Thr Leu Val Thr Leu Leu Val Glu AsnGly 210 215 220 Ala Asp Val Gln Ala Ala Ala Asn Gly Asp Phe Phe Lys LysThr Lys 225 230 235 240 Gly Arg Pro Gly Phe Tyr Phe Gly Glu Leu Pro LeuSer Leu Ala Ala 245 250 255 Cys Thr Asn Gln Leu Ala Ile Val Lys Phe LeuLeu Gln Asn Ser Trp 260 265 270 Gln Pro Ala Asp Ile Ser Ala Arg Asp SerVal Gly Asn Thr Val Leu 275 280 285 His Ala Leu Val Glu Val Ala Asp AsnThr Val Asp Asn Thr Lys Phe 290 295 300 Val Thr Ser Met Tyr Asn Glu IleLeu Ile Leu Gly Ala Lys Leu His 305 310 315 320 Pro Thr Leu Lys Leu GluGlu Ile Thr Asn Arg Lys Gly Leu Thr Pro 325 330 335 Leu Ala Leu Ala AlaSer Ser Gly Lys Ile Gly Val Leu Ala Tyr Ile 340 345 350 Leu Gln Arg GluIle His Glu Pro Glu Cys Arg His Leu Ser Arg Lys 355 360 365 Phe Thr GluTrp Ala Tyr Gly Pro Val His Ser Ser Leu Tyr Asp Leu 370 375 380 Ser CysIle Asp Thr Cys Glu Lys Asn Ser Val Leu Glu Val Ile Ala 385 390 395 400Tyr Ser Ser Ser Glu Thr Pro Asn Arg His Asp Met Leu Leu Val Glu 405 410415 Pro Leu Asn Arg Leu Leu Gln Asp Lys Trp Asp Arg Phe Val Lys Arg 420425 430 Ile Phe Tyr Phe Asn Phe Phe Val Tyr Cys Leu Tyr Met Ile Ile Phe435 440 445 Thr Ala Ala Ala Tyr Tyr Arg Pro Val Glu Gly Leu Pro Pro TyrLys 450 455 460 Leu Lys Asn Thr Val Gly Asp Tyr Phe Arg Val Thr Gly GluIle Leu 465 470 475 480 Ser Val Ser Gly Gly Val Tyr Phe Phe Phe Arg GlyIle Gln Tyr Phe 485 490 495 Leu Gln Arg Arg Pro Ser Leu Lys Ser Leu PheVal Asp Ser Tyr Ser 500 505 510 Glu Ile Leu Phe Phe Val Gln Ser Leu PheMet Leu Val Ser Val Val 515 520 525 Leu Tyr Phe Ser Gln Arg Lys Glu TyrVal Ala Ser Met Val Phe Ser 530 535 540 Leu Ala Met Gly Trp Thr Asn MetLeu Tyr Tyr Thr Arg Gly Phe Gln 545 550 555 560 Gln Met Gly Ile Tyr AlaVal Met Ile Glu Lys Met Ile Leu Arg Asp 565 570 575 Leu Cys Arg Phe MetPhe Val Tyr Leu Val Phe Leu Phe Gly Phe Ser 580 585 590 Thr Ala Val ValThr Leu Ile Glu Asp Gly Lys Asn Asn Ser Leu Pro 595 600 605 Met Glu SerThr Pro His Lys Cys Arg Gly Ser Ala Cys Lys Pro Gly 610 615 620 Asn SerTyr Asn Ser Leu Tyr Ser Thr Cys Leu Glu Leu Phe Lys Phe 625 630 635 640Thr Ile Gly Met Gly Asp Leu Glu Phe Thr Glu Asn Tyr Asp Phe Lys 645 650655 Ala Val Phe Ile Ile Leu Leu Leu Ala Tyr Val Ile Leu Thr Tyr Ile 660665 670 Leu Leu Leu Asn Met Leu Ile Ala Leu Met Gly Glu Thr Val Asn Lys675 680 685 Ile Ala Gln Glu Ser Lys Asn Ile Trp Lys Leu Gln Arg Ala IleThr 690 695 700 Ile Leu Asp Thr Glu Lys Ser Phe Leu Lys Cys Met Arg LysAla Phe 705 710 715 720 Arg Ser Gly Lys Leu Leu Gln Val Gly Phe Thr ProAsp Gly Lys Asp 725 730 735 Asp Tyr Arg Trp Cys Phe Arg Val Asp Glu ValAsn Trp Thr Thr Trp 740 745 750 Asn Thr Asn Val Gly Ile Ile Asn Glu AspPro Gly Asn Cys Glu Gly 755 760 765 Val Lys Arg Thr Leu Ser Phe Ser LeuArg Ser Gly Arg Val Ser Gly 770 775 780 Arg Asn Trp Lys Asn Phe Ala LeuVal Pro Leu Leu Arg Asp Ala Ser 785 790 795 800 Thr Arg Asp Arg His AlaThr Gln Gln Glu Glu Val Gln Leu Lys His 805 810 815 Tyr Thr Gly Ser LeuLys Pro Glu Asp Ala Glu Val Phe Lys Asp Ser 820 825 830 Met Val Pro GlyGlu Lys 835 <210> SEQ ID NO 10 <211> LENGTH: 502 <212> TYPE: DNA <213>ORGANISM: Rattus sp. <400> SEQUENCE: 10 agatcttgat cctgggggcc aaactccaccccacgctgaa gctggaagag atcaccaaca 60 ggaaggggct cacgccactg gctctggctgctagcagtgg gaagatcggg gtcttggcct 120 acattctcca gagggagatc catgaacccgagtgccgaca cctatccagg aagttcaccg 180 aatgggccta tgggccagtg cactcctccctttatgacct gtcctgcatt gacacctgtg 240 aaaagaactc ggttctggag gtgatcgcttacagcagcag tgagacccct aaccgtcatg 300 acatgcttct cgtggaaccc ttgaaccgactcctacagga caagtgggac agatttgtca 360 agcgcatctt ctacttcaac ttcttcgtctactgcttgta tatgatcatc ttcaccgcgg 420 ctgcctacta tcggcctgtg gaaggcttgcccccctataa gctgaaaaac accgttgggg 480 actatttccg agtcaccgga ga 502 <210>SEQ ID NO 11 <211> LENGTH: 819 <212> TYPE: DNA <213> ORGANISM: Rattussp. <400> SEQUENCE: 11 ccatgggctg gaccaacatg ctctactata cccgaggattccagcagatg ggcatctatg 60 ctgtcatgat tgagaagatg atcctcagag acctgtgccggtttatgttc gtctacctcg 120 tgttcttgtt tggattttcc acagctgtgg tgacactgattgaggatggg aagaataact 180 ctctgcctat ggagtccaca ccacacaagt gccgggggtctgcctgcaag ccaggtaact 240 cttacaacag cctgtattcc acatgtctgg agctgttcaagttcaccatc ggcatgggcg 300 acctggagtt cactgagaac tacgacttca aggctgtcttcatcatcctg ttactggcct 360 atgtgattct cacctacatc cttctgctca acatgctcattgctctcatg ggtgagaccg 420 tcaacaagat tgcacaagag agcaagaaca tctggaagctgcagagagcc atcaccatcc 480 tggatacaga gaagagcttc ctgaagtgca tgaggaaggccttccgctct ggcaagctgc 540 tgcaggtggg gttcactcct gacggcaagg atgactaccggtggtgtttc agggtggacg 600 aggtaaactg gactacctgg aacaccaatg tgggtatcatcaacgaggac ccaggcaact 660 gtgagggcgt caagcgcacc ctgagcttct ccctgaggtcaggccgagtt tcagggagaa 720 actggaagaa ctttgccctg gttccccttc tgagggatgcaagcactcga gatagacatg 780 ccacccagca ggaagaagtt caactgaagc attatacgg 819<210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Rattus sp. <400> SEQUENCE: 12 ctggaaagga tggaacaacg 20 <210> SEQ ID NO13 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Rattus sp. <400>SEQUENCE: 13 gctctagata ggaatgtgcc taggcagg 28

What is claimed is:
 1. An isolated nucleic acid molecule encoding theamino acid sequence of SEQ ID NO:4.
 2. An isolated nucleic acid moleculeencoding the amino acid sequence of SEQ ID NO:5.
 3. A nucleic acidvector for recombinant expression of a human capsaicin receptorcomprising a nucleic acid sequence encoding the amino acid sequence ofSEQ ID NO:4 operatively linked to a nucleic acid sequence comprising atleast one heterologous regulatory element in the appropriate orientationfor expression.
 4. The vector of claim 3, wherein the vector is aplasmid vector.
 5. The vector of claim 3, wherein the vector is a viralvector.
 6. A recombinant cell comprising the vector of claim 3, saidrecombinant cell obtained by introducing the vector of claim 3 into ahost cell that does not contain the vector of claim
 3. 7. Therecombinant cell of claim 6, wherein the recombinant cell is a bacterialcell.
 8. The recombinant cell of claim 6, wherein the recombinant cellexhibits capsaicin agonist binding activity that significantly greaterto the p<0.05 level, as measured using a parametric test of statisticalsignificance, than that exhibited by the host cell.
 9. The recombinantcell of claim 8, wherein the recombinant cell is an insect cell.
 10. Therecombinant cell of claim 8 wherein the cell is an amphibian cell. 11.The recombinant cell of claim 10 wherein the amphibian cell is anoocyte.
 12. The recombinant cell of claim 8, wherein the cell is amammalian cell.
 13. An assay for determining if a compound binds tocapsaicin receptors, which assay comprises: (a) contacting a firstplurality of the recombinant cell of claim 8 with the compound and alabeled capsaicin agonist to yield contacted test cells; (b) contactinga second plurality of the recombinant cell of claim 8 with the labeledcapsaicin agonist to yield control cells; (c) removing unbound labeledcapsaicin agonist from the contacted test cells of (a) to yield washedtest cells; (d) removing unbound labeled capsaicin agonist from thecontrol cells of (b) to yield washed control cells; (e) measuring theconcentration of label associated with the washed test cells of (c); (f)measuring the concentration of label associated with the washed controlcells of (d); (g) comparing the concentration of label associated withthe washed test cells of (c), as determined in (e) with theconcentration of label associated with the washed control cells of (d),as determined in (f); wherein a compound for which the comparison of (g)indicates that the concentration of label associated with the washedtest cells of (c), as determined in (e), is lower than concentration oflabel associated with the washed control cells of (d), as determined in(f), is a compound that binds to capsaicin receptors.
 14. An assay fordetermining if a compound is an agonist of capsaicin receptors, whichassay comprises: (a) contacting a plurality of the recombinant cell ofclaim 8 with an indicator of intracellular Ca²⁺ concentration to yieldindicator-loaded cells; (b) removing the excess indicator ofintracellular Ca²⁺ concentration from the indicator-loaded cells of (a)to yield washed indicator-loaded cells; (c) contacting the washedindicator-loaded cells of (b) with the compound to yield contacted testcells; (d) measuring detectable calcium levels in the contacted testcells of (c) by quantifying Ca²⁺-concentration-dependant changes in theproperties of the indicator of intracellular Ca²⁺ concentration in thecontacted test cells of (c); (e) measuring detectable calcium levels inthe washed indicator-loaded cells of (b) by quantifyingCa²⁺-concentration-dependant changes in the properties of the indicatorof intracellular Ca²⁺ concentration in the washed indicator-loaded cellsof (b); (f) comparing detectable calcium levels in the contacted testcells of (c), as measured in (d), with detectable calcium levels in thewashed indicator-loaded cells of (b), as measured in (e); wherein a testcompound for which the comparison of (f) demonstrates that thedetectable calcium levels in the contacted test cells of (c) is at least2-fold greater than the detectable calcium levels in the washedindicator-loaded cells of (d) is an agonist of capsaicin receptors. 15.An assay for determining if a compound is an antagonist of capsaicinreceptors, which assay comprises: (a) contacting a plurality of therecombinant cell of claim 8 with the compound and an indicator ofintracellular Ca²⁺ concentration to yield contacted test cells; (b)contacting a plurality of the recombinant cell of claim 8 with theindicator of intracellular Ca²⁺ concentration to yield control cells;(c) removing the excess indicator of intracellular Ca²⁺ concentrationfrom the control cells of (a) to yield washed test cells; (d) removingthe excess indicator of intracellular Ca²⁺ concentration from thecontacted test cells of (b) to yield washed control cells; (e) adding anagonist of the capsaicin receptor to the washed test cells of (c) toyield agonist-contacted test cells; (f) adding the agonist of thecapsaicin receptor to the washed test cell of (d) to yieldagonist-contacted control cells; (g) measuring the concentration ofintracellular Ca²⁺in the agonist-contacted test cells of (e) bymeasuring changes in the properties of the indicator of intracellularCa²⁺ concentration; (h) measuring the concentration of intracellularCa²⁺ in the agonist-contacted control cells of (f) by measuring changesin the properties of the indicator of intracellular Ca²⁺ concentration;(i) comparing the concentration of intracellular Ca²⁺ in theagonist-contacted test cells of (e), as measured in (g) with theconcentration of intracellular Ca²⁺ in the agonist-contacted controlcells of (f), as measured in (h); wherein a test compound for which thecomparison of (i) indicates that the concentration of intracellular Ca²⁺in the agonist-contacted test cells of (e) is significantly less to thep≦0.05 level, as measured using a parametric test of statisticalsignificance, than the concentration of intracellular Ca²⁺ in theagonist-contacted control cells of (f), is an antagonist of capsaicinreceptors.
 16. A membrane preparation comprising isolated membranes of aplurality of the recombinant cell of claim 8, wherein the isolatedmembranes exhibit capsaicin receptor agonist binding activity that issignificantly greater to the p≦0.05 level, as measured using aparametric test of statistical significance, than that exhibited bymembranes isolated from a plurality of the host cells.
 17. An assay fordetermining if a compound binds to capsaicin receptors, which assaycomprises: (a) contacting a first aliquot of the membrane preparation ofclaim 16 with the compound and a labeled capsaicin agonist to yieldcontacted test membranes; (b) contacting a second aliquot of themembrane preparation of claim 16 with a labeled capsaicin agonist toyield control membranes; (c) removing unbound labeled capsaicin agonistfrom the contacted test membranes of (a) to yield washed test membranes;(d) removing unbound labeled capsaicin agonist from the controlmembranes of (b) to yield washed control membranes; (e) measuring theconcentration of label associated with the washed test membranes of (c);(f) measuring the concentration of label associated with the washedcontrol membranes of (d) (g) comparing the concentration of labelassociated with the washed test membranes of (c), as determined in (e),with the concentration of label associated with the washed controlmembranes of (d), as determined in (f); wherein a test compound forwhich the comparison of (g) indicates that the concentration of labelassociated with the washed test membranes of (c) is lower thanconcentration of label associated with the washed control membranes of(d), is a compound that binds to capsaicin receptors.
 18. An isolatedcell expressing at least 1.5×10⁵ capsaicin receptor ligand binding sitesper cell.
 19. A culture of the cells of claim
 18. 20. The cell of claim18, wherein the cell is a mammalian cell.
 21. The cell of claim 18,wherein the cell expresses at least 1.5×10⁶ capsaicin receptor ligandbinding sites per cell.
 22. The cell of claim 18, wherein the cellexpresses at least 3×10⁶ capsaicin receptor ligand binding sites percell.
 23. An isolated cell expressing capsaicin receptors at a level ofat least 200 fmol of capsaicin receptors per 10⁶ cells.
 24. A culture ofthe cells of claim
 23. 25. The cell of claim 23, wherein the cellexpresses at least 450 fmol of capsaicin receptor per 10⁶ cells. 26.Isolated cell membranes comprising at least 2.35 pmol of capsaicinreceptor per mg of total membrane protein.
 27. The isolated cellmembranes of claim 26, wherein the membranes comprise at least 4.2 pmol,of capsaicin receptor per mg of total membrane protein.
 28. The isolatedcell membranes of claim 26, wherein the membranes comprise at least 25pmol, of capsaicin receptor per mg of total membrane protein.
 29. Anassay for determining if a compound binds to capsaicin receptors, whichassay comprises: (a) contacting a first plurality of the cell of claim18 with the compound and a labeled capsaicin agonist to yield contactedtest cells; (b) contacting a second plurality of the recombinant cell ofclaim 18 with a labeled capsaicin agonist to yield control cells; (c)removing unbound labeled capsaicin agonist from the contacted test cellsof (a) to yield washed test cells; (d) removing unbound labeledcapsaicin agonist from the control cells of (b) to yield washed controlcells; (e) measuring the concentration of label associated with thewashed test cells of (c); (f) measuring the concentration of labelassociated with the washed control cells of (d); (g) comparing theconcentration of label associated with the washed test cells of (c), asdetermined in (e), with the concentration of label associated with thewashed control cells of (d), as determined in (f); wherein a testcompound for which the comparison of (g) indicates that theconcentration of label associated with the washed test cells of (c), asdetermined in (e), is lower than concentration of label associated withthe washed control cells of (d), as determined in (f), binds tocapsaicin receptors.
 30. An assay for determining if a compound is anagonist of capsaicin receptors, which comprises: (a) contacting aplurality of the recombinant cell of claim 18 with an indicator ofintracellular Ca²⁺ concentration to yield indicator-loaded cells; (b)removing the excess indicator of intracellular Ca²⁺ concentration fromthe indicator-loaded cells of (a) to yield washed indicator-loadedcells; (c) contacting the washed indicator-loaded cells of (b) with thecompound to yield contacted test cells; (d) measuring detectable calciumlevels in the contacted test cells of (c) by quantifyingCa²⁺-concentration-dependant changes in the properties of the indicatorof intracellular Ca²⁺ concentration in the contacted test cells of (c);(e) measuring detectable calcium levels in the washed indicator-loadedcells of (b) by quantifying Ca²⁺-concentration-dependant changes in theproperties of the indicator of intracellular Ca²⁺ concentration in thewashed indicator-loaded cells of (b); (f) comparing detectable calciumlevels in the contacted test cells of (c), as measured in (d), withdetectable calcium levels in the washed indicator-loaded cells of (b),as measured in (e); wherein a test compound for which the comparison of(f) demonstrates that the detectable calcium levels in the contactedtest cells of (c) is at least 2-fold greater than the detectable calciumlevels in the washed indicator-loaded cells of (d) is an agonist ofcapsaicin receptors.
 31. An assay for determining if a compound is anantagonist of capsaicin receptors, which comprises: (a) contacting aplurality of the recombinant cell of claim 18 with the compound and anindicator of intracellular Ca²⁺ concentration to yield contacted testcells; (b) contacting a plurality of the recombinant cell of claim 18with the indicator of intracellular Ca²⁺ concentration to yield controlcells; (c) removing the excess indicator of intracellular Ca²⁺concentration from the control cells of (a) to yield washed test cells;(d) removing the excess indicator of intracellular Ca²⁺ concentrationfrom the contacted test cells of (b) to yield washed control cells; (e)adding an agonist of the capsaicin receptor to the washed test cells of(c) to yield agonist-contacted test cells; (f) adding the agonist of thecapsaicin receptor to the washed test cell of (d) to yieldagonist-contacted control cells; (g) measuring the concentration ofintracellular Ca²⁺ in the agonist-contacted test cells of (e) bymeasuring changes in the properties of the indicator of intracellularCa²⁺ concentration; (h) measuring the concentration of intracellularCa²⁺ in the agonist-contacted control cells of (f) by measuring changesin the properties of the indicator of intracellular Ca²⁺ concentration;(i) comparing the concentration of intracellular Ca²⁺ in theagonist-contacted test cells of (e), as measured in (g) with theconcentration of intracellular Ca²⁺ in the agonist-contacted controlcells of (f), as measured in (h); wherein a test compound for which thecomparison of (i) indicates that the concentration of intracellular Ca²⁺in the agonist-contacted test cells of (e) is significantly less to thep≦0.05 level, as measured using a parametric test of statisticalsignificance, than the concentration of intracellular Ca²⁺ in theagonist-contacted control cells of (f), is an antagonist of capsaicinreceptors.
 32. An assay for determining if a compound binds to capsaicinreceptors, which comprises: (a) contacting a first aliquot of theisolated cell membranes of claim 27 with the compound and a labeledcapsaicin agonist to yield contacted test membranes; (b) contacting asecond aliquot of the isolated cell membranes of claim 27 with a labeledcapsaicin agonist to yield control membranes; (c) removing unboundlabeled capsaicin agonist from the contacted test membranes of (a) toyield washed test membranes; (d) removing unbound labeled capsaicinagonist from the control membranes of (b) to yield washed controlmembranes; (e) measuring the concentration of label associated with thewashed test membranes of (c); (f) measuring the concentration of labelassociated with the washed control membranes of (d) (g) comparing theconcentration of label associated with the washed test membranes of (c),as determined in (e), with the concentration of label associated withthe washed control membranes of (d), as determined in (f); wherein atest compound for which the comparison of (g) indicates that theconcentration of label associated with the washed test membranes of (c)is lower than concentration of label associated with the washed controlmembranes of (d), is a compound that binds to capsaicin receptors.
 33. Avector for recombinant expression of a polypeptide with an amino acidsequence consisting essentially of the amino acid sequence of SEQ ID NO:5, said vector comprising the nucleic acid molecule of claim 2operatively linked to at least one heterologous regulatory element inthe appropriate orientation for expression.
 34. The vector of claim 33wherein the at least one heterologous regulatory element is adapted forexpression in a bacterial host.
 35. A bacterial cell comprising thevector of claim 34, whereby the bacterial cell expresses a polypeptidewith an amino acid sequence consisting essentially of the amino acidsequence of SEQ ID NO:
 5. 36. An isolated polypeptide comprising anamino acid sequence corresponding to amino acids 2 (Lys) to 511 (Gln) ofSEQ ID NO:5.
 37. An isolated polypeptide comprising an amino acidsequence corresponding to amino acids 2 (Lys) to 839 (Lys) of SEQ IDNO:4.
 38. A cell that has been stably transfected with a nucleic acidvector for recombinant expression of a capsaicin receptor, said vectorcomprising a sequence encoding a polypeptide exhibiting capsaicinreceptor ligand binding activity operatively linked to at least oneheterologous regulatory element sequence in the appropriate orientationfor expression.
 39. Plasmid vector PT35, having ATCC patent depositnumber PTA-576.
 40. Plasmid vector PT44, having ATCC patent depositnumber PTA-577.
 41. Plasmid vector PT36, having ATCC patent depositnumber PTA-578.